Covalent tethering of functional groups to proteins and substrates therefor

ABSTRACT

A mutant hydrolase optionally fused to a protein of interest is provided. The mutant hydrolase is capable of forming a bond with a substrate for the corresponding nonmutant (wild-type) hydrolase which is more stable than the bond formed between the wild-type hydrolase and the substrate and has at least two amino acid substitutions relative to the wild-type hydrolase. Substrates for hydrolases comprising one or more functional groups are also provided, as well as methods of using the mutant hydrolase and the substrates of the invention. Also provided is a fusion protein capable of forming a stable bond with a substrate and cells which express the fusion protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/895,119, filed May 15, 2013, which is a divisional of U.S. patentapplication Ser. No. 13/435,970, filed Mar. 30, 2012, which is adivisional of U.S. Pat. No. 8,168,405, issued May 1, 2012, which is adivisional of U.S. Pat. No. 7,935,803, issued May 3, 2011, which is adivisional of U.S. Pat. No. 7,425,436, issued Sep. 16, 2008, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/592,499,filed Jul. 30, 2004 and is a continuation-in-part of U.S. Pat. No.7,429,472, issued Sep. 30, 2008, which claims the benefit of U.S.Provisional Application Ser. No. 60/592,499, filed Jul. 30, 2004, eachof which are incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to the field of biochemical assays and reagents.More specifically, this invention relates to mutant proteins covalentlylinked (tethered) to one or more functional groups and to methods fortheir use.

BACKGROUND OF THE INVENTION

The specific detection of molecules is a keystone in understanding therole of that molecule in the cell. Labels, e.g., those that arecovalently linked to a molecule of interest, permit the ready detectionof that molecule in a complex mixture. The label may be one that isadded by chemical synthesis in vitro or attached in vivo, e.g., viarecombinant techniques. For instance, the attachment of fluorescent orother labels onto proteins has traditionally been accomplished by invitro chemical modification after protein purification (Hermanson,1996). For in vivo attachment of a label, green fluorescent protein(GFP) from the jellyfish Aequorea victoria can be genetically fused withmany host proteins to produce fluorescent chimeras in situ (Tsien, 1998;Chalfie et al., 1998). However, while GFP-based indicators are currentlyemployed in a variety of assays, e.g., measuring pH (Kneen et al., 1998;Llopis et al., 1998; Miesenböck et al., 1998), Ca²⁺ (Miyawaki et al.,1997; Rosomer et al., 1997), and membrane potential (Siegel et al.,1997), the fluorescence of intrinsically labeled proteins such as GFP islimited by the properties of protein structure, e.g., a limited range offluorescent colors and relatively low intrinsic brightness (Cubitt etal., 1995; Ormö et al., 1996).

To address the deficiencies of GFP labeling in situ, Griffen et al.(1998) synthesized a tight-binding pair of molecular components: a smallreceptor domain composed of as few as six natural amino acids and asmall (<700 dalton), synthetic ligand that could be linked to variousspectroscopic probes or crosslinks. The receptor domain included fourcysteines at the i, i+1, i+4, and i+5 positions of an α helix and theligand was 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FLASH).Griffen et al. disclose that the ligand had relatively few binding sitesin nontransfected mammalian cells, was membrane-permeant and wasnonfluorescent until it bound with high affinity and specificity to atetracysteine domain in a recombinant protein, resulting in cells beingfluorescently labeled (“FLASH” labeled) with a nanomolar or lowerdissociation constant. However, with respect to background binding incells, Stroffekova et al. (2001) disclose that FLASH-EDT₂ bindsnon-specifically to endogenous cysteine-rich proteins. Furthermore,labeling proteins by FLASH is limited by the range of fluorophores thatmay be used.

Receptor-mediated targeting methods use genetically encoded targetingsequences to localize fluorophores to virtually any cellular site,provided that the targeted protein is able to fold properly. Forexample, Farinas et al. (1999) disclose that cDNA transfection was usedto target a single-chain antibody (sFv) to a specified site in a cell.Farinas et al. disclose that conjugates of a hapten(4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, phOx) and a fluorescentprobe (e.g., BODIPY Fl, tetramethylrhodamine, and fluorescein) werebound with high affinity (about 5 nM) to the subcellular site for thesFv in living Chinese hamster ovary cells, indicating that the targetedantibody functioned as a high affinity receptor for the cell-permeablehapten-fluorophore conjugates. Nevertheless, functional sFv expressionmay be relatively poor in reducing environments.

Thus, what is needed is an improved method to label a desired molecule.

SUMMARY OF THE INVENTION

The invention provides methods, compositions and kits for tethering(linking), e.g., via a covalent or otherwise stable bond, one or morefunctional groups to a protein of the invention or to a fusion protein(chimera) which includes a protein of the invention. A protein of theinvention is structurally related to a wild-type (native) hydrolase butincludes at least one amino acid substitution, and in some embodimentsat least two amino acid substitutions, relative to the correspondingwild-type hydrolase, and binds a substrate of the correspondingwild-type hydrolase but lacks or has reduced catalytic activity relativeto the corresponding wild-type hydrolase (which mutant protein isreferred to herein as a mutant hydrolase). The aforementioned tetheringoccurs, for instance, in solution or suspension, in a cell, on a solidsupport or at solution/surface interfaces, by employing a substrate fora hydrolase which includes a reactive group and which has been modifiedto include one or more functional groups. As used herein, a “substrate”includes a substrate having a reactive group and optionally one or morefunctional groups. A substrate which includes one or more functionalgroups is generally referred to herein as a substrate of the invention.As used herein, a “functional group” is a molecule which is detectableor is capable of detection, for instance, a molecule which is measurableby direct or indirect means (e.g., a photoactivatable molecule,digoxigenin, nickel NTA (nitrilotriacetic acid), a chromophore,fluorophore or luminophore), can be bound or attached to a secondmolecule (e.g., biotin, hapten, or a cross-linking group), or may be asolid support.

A functional group may have more than one property such as being capableof detection and of being bound to another molecule. As used herein a“reactive group” is the minimum number of atoms in a substrate which arespecifically recognized by a particular wild-type or mutant hydrolase ofthe invention. The interaction of a reactive group in a substrate and awild-type hydrolase results in a product and the regeneration of thewild-type hydrolase. A substrate, e.g., a substrate of the invention,may also optionally include a linker, e.g., a cleavable linker, whichphysically separates one or more functional groups from the reactivegroup in the substrate, and in one embodiment, the linker is preferably12 to 30 atoms in length. The linker may not always be present in asubstrate of the invention, however, in some embodiments, the physicalseparation of the reactive group and the functional group may be neededso that the reactive group can interact with the reactive residue in themutant hydrolase to form a covalent bond. Preferably, when present, thelinker does not substantially alter, e.g., impair, the specificity orreactivity of a substrate having the linker with the wild-type or mutanthydrolase relative to the specificity or reactivity of a correspondingsubstrate which lacks the linker with the wild-type or mutant hydrolase.Further, the presence of the linker preferably does not substantiallyalter, e.g., impair, one or more properties, e.g., the function, of thefunctional group. For instance, for some mutant hydrolases, i.e., thosewith deep catalytic pockets, a substrate of the invention can include alinker of sufficient length and structure so that the one or morefunctional groups of the substrate of the invention do not disturb the3-D structure of the hydrolase (wild-type or mutant). For example, oneexample of a substrate of the invention for a dehalogenase includes areactive group such as (CH₂)₂₋₃X where X is a halide and a functionalgroup such as carboxytetramethylrhodamine, e.g.,carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl.

In one embodiment, the invention provides a compound of formula (I):R-linker-A-X, wherein R is one or more functional groups, wherein thelinker is a multiatom straight or branched chain including C, N, S, orO, or a group that comprises one or more rings, e.g., saturated orunsaturated rings, such as one or more aryl rings, heteroaryl rings, orany combination thereof, wherein A-X is a substrate for a dehalogenase,e.g., a haloalkane dehalogenase or a dehalogenase that cleavescarbon-halogen bonds in an aliphatic or aromatic halogenated substrate,such as a substrate for Rhodococcus, Sphingomonas, Staphylococcus,Pseudomonas, Burkholderia, Agrobacterium or Xanthobacter dehalogenase,and wherein X is a halogen. In one embodiment, an alkylhalide iscovalently attached to a linker, L, which is a group or groups thatcovalently attach one or more functional groups to form a substrate fora dehalogenase. As described herein, a mutant of a Rhodococcusdehalogenase (DhaA) (see FIG. 2 for an exemplary wild-type Rhodococcusdehalogenase “DhaA.WT” sequence), DhaA.H272F, was bound to substratesfor DhaA which included 5-(and 6-) carboxyfluorescein, e.g.,carboxyfluorescein-C₁₀H₂₁NO₂—Cl, carboxytetramethylrhodamine, e.g.,carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl, and biotin, e.g.,biotin-C₁₀H₂₁NO₂—Cl, and there was no significant quenching effect ofthis binding on carboxyfluorescein or carboxytetramethylrhodaminefluorescence or on biotin binding to streptavidin. As also describedherein, a mutant dehalogenase, e.g., DhaA.D106C and DhaA.D106E as wellas DhaA.D106C:H272F and DhaA.D106E:H272F, boundcarboxyfluorescein-C₁₀H₂₁NO₂—Cl and/orcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl. In one embodiment, thesubstrate is R—(CH₂)₂O(CH₂)₂O(CH₂)₂O(CH₂)₆Cl, wherein R is a functionalgroup. To prepare such a substrate, a functional group may be reactedwith a molecule such as NH(CH₂)₂O(CH₂)₂O(CH₂)₂O(CH₂)₆Cl.

In one embodiment, substrates of the invention are permeable to theplasma membranes of cells. For instance, as described herein the plasmamembranes of prokaryotic (E. coli) and eukaryotic (CHO-K1) cells werepermeable to carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl andbiotin-C₁₀H₂₁NO₂—Cl and, these substrates were rapidly and efficientlyloaded into and washed out of cells in the absence of a mutanthydrolase. In the presence of a mutant hydrolase, at least a portion ofthe substrate was prevented from being washed out of the cells. Thus,the bound portion of the substrate can serve as a marker or as a meansto capture the mutant hydrolase or a fusion thereof.

In one embodiment, the substrate of the invention includes two or morefunctional groups. In one embodiment, one of the functional groups is anenzyme. In another embodiment, one of the functional groups is asubstrate for an enzyme. For example, one functional group may beluciferin and the other a protease recognition site, i.e., one whichcontains sequences sufficient for recognition by the protease includingthe site to be cleaved, one functional group may be biotin and the othera fluorophore, or one functional group may be a protease recognitionsite and the other a fluorophore.

The invention further provides methods for preparing a substrate for ahydrolase which substrate is modified to include one or more functionalgroups.

A mutant hydrolase of the invention, as described in more detail herein,comprises at least one amino acid substitution relative to acorresponding wild-type hydrolase, wherein the at least one amino acidsubstitution results in the mutant hydrolase forming a bond with thesubstrate which is more stable than the bond formed between thecorresponding wild-type hydrolase and the substrate. The at least oneamino acid substitution in the mutant hydrolase is a substitution at anamino acid residue in the corresponding wild-type hydrolase that isassociated with activating a water molecule which cleaves the bondformed between the corresponding wild-type hydrolase and the substrateor at an amino acid residue in the corresponding wild-type hydrolasethat forms an ester intermediate with the substrate. In one embodiment,the mutant hydrolase comprises at least two amino acid substitutionsrelative to a corresponding wild-type hydrolase, wherein onesubstitution is in a residue which, in the wild-type hydrolase, isassociated with activating a water molecule or in a residue which, inthe wild-type hydrolase, forms an ester intermediate by nucleophilicattack of a substrate for the hydrolase, and another substitution in aresidue which, in the wild-type hydrolase, is at or near a bindingsite(s) for a hydrolase substrate, e.g., the residue within 3 to 5 Å ofa hydrolase substrate bound to a wild-type hydrolase but is not in aresidue that in the corresponding wild-type hydrolase is associated withactivating a water molecule or which forms ester intermediate with asubstrate. In one embodiment, the second substitution is in a residuewhich, in the wild-type hydrolase lines the site(s) for substrate entryinto the catalytic pocket of the hydrolase, e.g., a residue that iswithin the active site cavity and within 3 to 5 Å of a hydrolasesubstrate bound to the wild-type hydrolase such as a residue in a tunnelfor the substrate that is not a residue in the corresponding wild-typehydrolase which is associated with activating a water molecule or whichforms an ester intermediate with a substrate. The additionalsubstitution(s) preferably increase the rate of stable covalent bondformation of those mutants binding to a substrate of a correspondingwild-type hydrolase.

The mutant hydrolase may be a fusion protein, e.g., a fusion proteinexpressed from a recombinant DNA which encodes the mutant hydrolase andat least one protein of interest or a fusion protein formed by chemicalsynthesis. For instance, the fusion protein may comprise a mutanthydrolase and an enzyme of interest, e.g., luciferase, RNasin or RNase,and/or a channel protein, a receptor, a membrane protein, a cytosolicprotein, a nuclear protein, a structural protein, a phosphoprotein, akinase, a signaling protein, a metabolic protein, a mitochondrialprotein, a receptor associated protein, a fluorescent protein, an enzymesubstrate, a transcription factor, a transporter protein and/or atargeting sequence, e.g., a myristilation sequence, a mitochondriallocalization sequence, or a nuclear localization sequence, that directsthe mutant hydrolase, for example, a fusion protein, to a particularlocation. The protein of interest may be fused to the N-terminus or theC-terminus of the mutant hydrolase. In one embodiment, the fusionprotein comprises a protein of interest at the N-terminus, and anotherprotein, e.g., a different protein, at the C-terminus, of the mutanthydrolase. For example, the protein of interest may be a fluorescentprotein or an antibody. Optionally, the proteins in the fusion areseparated by a connector sequence, e.g., preferably one having at least2 amino acid residues, such as one having 13 to 17 amino acid residues.The presence of a connector sequence in a fusion protein of theinvention does not substantially alter the function of either protein inthe fusion relative to the function of each individual protein.

Also provided is an isolated nucleic acid molecule (polynucleotide)comprising a nucleic acid sequence encoding a hydrolase, e.g., a mutanthydrolase of the invention. In one embodiment, the isolated nucleic acidmolecule comprises a nucleic acid sequence which is optimized forexpression in at least one selected host. Optimized sequences includesequences which are codon optimized, i.e., codons which are employedmore frequently in one organism relative to another organism, e.g., adistantly related organism, as well as modifications to add or modifyKozak sequences and/or introns, and/or to remove undesirable sequences,for instance, potential transcription factor binding sites. In oneembodiment, the polynucleotide includes a nucleic acid sequence encodinga dehalogenase, which nucleic acid sequence is optimized for expressionis a selected host cell. In one embodiment, the optimized polynucleotideno longer hybridizes to the corresponding non-optimized sequence, e.g.,does not hybridize to the non-optimized sequence under medium or highstringency conditions. In another embodiment, the polynucleotide hasless than 90%, e.g., less than 80%, nucleic acid sequence identity tothe corresponding non-optimized sequence and optionally encodes apolypeptide having at least 80%, e.g., at least 85%, 90% or more, aminoacid sequence identity with the polypeptide encoded by the non-optimizedsequence. Constructs, e.g., expression cassettes, and vectors comprisingthe isolated nucleic acid molecule, as well as kits comprising theisolated nucleic acid molecule, construct or vector are also provided.

The invention also includes compositions and kits comprising a substratefor a hydrolase which includes a linker, a substrate for a hydrolasewhich includes one or more functional groups and optionally a linker, alinker which includes one or more functional groups, a substrate for ahydrolase which lacks one or more functional groups and optionallyincludes a linker, a linker, or a mutant hydrolase, or any combinationthereof. For example, the invention includes a solid support comprisinga substrate of the invention, a solid support comprising a mutanthydrolase of the invention or a fusion thereof, a kit comprising asubstrate of the invention, a kit comprising a vector encoding adehalogenase of the invention or a fusion thereof, or a kit comprising avector encoding a serine beta-lactamase of the invention or a fusionthereof.

The substrates and mutant hydrolases of the invention are useful toisolate, detect, identify, image, display, or localize molecules ofinterest, label cells, including live cell imaging, or label proteins invitro and/or in vivo. For instance, a substrate of the invention boundto a solid support or a mutant hydrolase bound to a solid support may beused to generate protein arrays, cell arrays, vesicle/organelle arrays,gene arrays, and/or cell membrane arrays. Thus, in one embodiment, theinvention provides a method to isolate a molecule of interest. Themethod includes providing a sample comprising one or more fusionproteins at least one of which comprises a mutant hydrolase of theinvention and a protein which is bound to the molecule of interest, anda solid support comprising one or more hydrolase substrates. The sampleand the solid support are then contacted so as to isolate the moleculeof interest. For instance, the method may be employed to isolate DNAbound to a protein fused to a mutant hydrolase.

In one embodiment, the invention provides a method to detect ordetermine the presence or amount of a mutant hydrolase. The methodincludes contacting a mutant hydrolase of the invention with a hydrolasesubstrate which comprises one or more functional groups. The presence oramount of the functional group is detected or determined, therebydetecting or determining the presence or amount of the mutant hydrolase.In one embodiment, the mutant hydrolase is in or on the surface of acell. In another embodiment, the mutant hydrolase is in a cell lysate.

Also provided are methods of using a mutant hydrolase of the inventionand a substrate for a corresponding hydrolase which includes one or morefunctional groups, e.g., to isolate a molecule or to detect or determinethe presence or amount of, location, e.g., intracellular, subcellular orextracellular location, or movement of certain molecules in cells.

In another embodiment, the invention includes a method to identify anagent that alters the interaction of a protein of interest with amolecule suspected of interacting with the protein of interest. Themethod includes contacting at least one agent with the moleculesuspected of interacting with the protein of interest, a fusion proteincomprising mutant hydrolase of the invention and the protein ofinterest, and a hydrolase substrate which comprises one or morefunctional groups. Then it is determined whether the agent alters theinteraction between the protein of interest and the molecule suspectedof interacting with the protein of interest.

The invention thus provides methods to monitor the expression, locationand/or movement (trafficking) of proteins in a cell as well as tomonitor changes in microenvironments within a cell. In one embodiment,the use of a mutant hydrolase of the invention and a substrate of theinvention permits functional analysis of proteins, e.g., ion channels.In another embodiment, the use of two pairs of a mutanthydrolase/substrate permits multiplexing, simultaneous detection, andFRET- or BRET-based assays.

To isolate, sort or purify cells, a mutant hydrolase of the inventionmay be expressed on the outside surface of cells (e.g., via a fusionwith a plasma membrane protein or a membrane anchoring signal). Forinstance, cells which express a fusion of a cytoplasmic andtransmembrane domains of an integrin with a mutant hydrolase, or afusion of a glycosylphosphatidyl inositol signal sequence and a mutanthydrolase, may be isolated (“captured”) by contacting those cells with asubstrate of the invention, for instance, one bound to a solid support.To isolate, purify or separate organelles, the mutant hydrolase isexpressed on the cytosolic surface of the organelle of interest. Inanother embodiment, to create an optimal platform for growing differentcells, the mutant hydrolase is fused with an extracellular matrixcomponent or an outer membrane protein and tethered to athree-dimensional cell culture or a platform for tissue engineering. Asan example, primary neurons or embryonic stem cells may be grown on theplatform to form a feeder layer.

Other applications include detecting or labeling cells. Thus, the use ofa mutant hydrolase of the invention and a corresponding substrate of theinvention permits the detection of cells, for instance, to detect cellmigration in vitro or in vivo after implantation or injection intoanimals (e.g., angiogenesis/chemotaxis assays, migration of implantedneurons, normal, malignant, or recombinantly modified cellsimplanted/injected into animals, and the like), and live cell imagingfollowed by immunocytochemistry. In another embodiment, the inventionprovides a method to label newly synthesized proteins. For example,cells comprising a vector which expresses a mutant hydrolase of theinvention or a fusion thereof, are contacted with a substrate for thehydrolase which lacks a functional group. Cells are then contacted withan agent, e.g., an inducer of gene expression, and a substrate for thehydrolase which contains one or more functional groups. The presence,amount or location of the mutant hydrolase or fusion thereof is thendetected or determined. The presence, amount or location of the mutanthydrolase or fusion thereof is due to newly synthesized mutant hydrolaseor a fusion thereof. Alternatively, cells comprising a vector whichexpresses a mutant hydrolase of the invention or a fusion thereof, arecontacted with a substrate for the hydrolase having a functional group,e.g., a green fluorophore, then contacted with an agent and a substratehaving a different functional group, e.g., a red fluorophore. In oneembodiment, the mutant hydrolase is fused to a membrane localizationsignal and so can be employed to monitor events in or near the membrane.

The invention also provides a method to label a cell, e.g., in atransgenic or non-transgenic non-human animal. For instance, to labelcells, the mutant hydrolase may be expressed on the outside surface ofcells (e.g., via a fusion with a plasma membrane protein or a membraneanchoring signal). For instance, cells which express a fusion of acytoplasmic and transmembrane domains of an integrin with a mutanthydrolase of the invention, or a fusion of a glycosylphosphatidylinositol signal sequence and a mutant hydrolase of the invention, may beidentified or labeled by contacting those cells with a substrate of theinvention. In one embodiment, the invention includes a method to labelcells in a transgenic animal. The method includes providing a transgenicnon-human animal, the genome of cells of which is augmented with anexpression cassette comprising a transcriptional regulatory elementwhich is optionally tissue- or cell-specific operably linked to nucleicacid fragment encoding a mutant hydrolase of the invention andoptionally a targeting peptide. The transgenic non-human animal is thencontacted with a hydrolase substrate that comprises one or morefunctional groups, thereby labeling cells that express the mutanthydrolase.

Cells expressing selectable marker proteins, such as ones encodingresistance to neomycin, hygromycin, or puromycin, are used to stablytransform cells with foreign DNA. It may be desirable to observe whichcells contain selectable marker proteins as well as fluorescentlylabeled molecules. For instance, it may be preferable to label theselectable marker protein with a fluorescent molecule that is addedexogenously to living cells. By this method, the selectable markerprotein becomes visible when only when needed by addition of thefluorophore, and the fluorescence will subsequently be lost whenselectable marker proteins are naturally regenerated through cellularmetabolism. Thus, in one embodiment, the invention provides a method forlabeling a cell which expresses a selectable marker protein. The methodincludes providing a cell comprising an expression cassette comprising anucleic acid sequence encoding a fusion protein. The fusion proteincomprises a selectable marker protein, e.g., one which confersresistance to at least one antibiotic, and a second protein that iscapable of stably and optionally irreversibly binding a substrate or aportion thereof which includes an optically detectable molecule. Forinstance, the protein may be an alkyl transferase which irreversiblytransfers an alkyl group and an optically detectable molecule from asubstrate to itself, thereby labeling the alkyl transferase, e.g., analkyl transferase such as O⁶-alkylguanine DNA alkyltransferase.Exemplary proteins useful in this embodiment of the invention include,but are not limited to, alkyl transferases, peptidylglycine-alpha-amidating monoxygenases, type I topoisomerases,hydrolases, e.g., serine and epoxide hydrolases as well as the mutanthydrolases described herein, aminotransferases, cytochrome P450monooxygenases, acetyl transferases, decarboxylases, oxidases, e.g.,monoamine oxidases, reductases, e.g., ribonucleotide reductase,synthetases, e.g., cyclic ADP ribose synthetase or thymidylatesynthetase, dehydrogenases, e.g., aldehyde dehydrogenase, synthases,e.g., nitric oxide synthase (NOS), lactamases, cystathioninegamma-lyases, peptidases, e.g., carboxypeptidase A, aromatase,proteases, e.g., serine protease, xylanases, glucosidases, mannosidases,and demethylases and other proteins, including wild-type proteins, whichform an irreversible or otherwise stable bond with one or moresubstrates, e.g., enzymes which are capable of mechanism-basedinactivation. Thus, in this embodiment, a stable bond, i.e., one whichis formed between a substrate and a wild-type or mutant enzyme, has at_(1/2) of at least 30 minutes and preferably at least 4 hours, and upto at least 10 hours, and is resistant to disruption by washing, proteindenaturants, and/or high temperatures, e.g., the bond is stable toboiling in SDS.

The cell which expresses the fusion protein is contacted with thesubstrate so as to label the cell. In one embodiment, the cell is fixedprior to contact with the substrate. In another embodiment, thesubstrate and fixative are contacted with the cell at the same time. Inyet another embodiment, the fixative is added to the cell after the cellis contacted with the substrate. In one embodiment, the fusion proteinforms an ester bond with the substrate. In another embodiment, thefusion protein forms a thioester bond with the substrate.

The invention also provides processes and intermediates disclosed hereinthat are useful for preparing compounds, compositions, nucleic acids,proteins, or other materials of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B provide a schematic of the two-step catalytic mechanism ofDhaA with an alkylhalide substrate. A). Nucleophilic displacement of ahalide group by Asp106 carboxylate and the formation of a covalent esterintermediate. B). Hydrolysis of the covalent intermediate by anactivated water molecule releasing alcohol and regenerating thecatalytic Asp106.

FIG. 2A shows a molecular model of the DhaA.H272F protein. The helicalcap domain is shown in light blue. The α/β hydrolase core domain (darkblue) contains the catalytic triad residues. The red shaded residuesnear the cap and core domain interface represent H272F and the D106nucleophile. The yellow shaded residues denote the positions of E130 andthe halide-chelating residue W107.

FIG. 2B shows the sequence of a Rhodococcus rhodochrous dehalogenase(DhaA) protein (Kulakova et al., 1997) (SEQ ID NO:82). The catalytictriad residues Asp (D), Glu (E) and His (H) are underlined. The residuesthat make up the cap domain are shown in italics. The DhaA.H272F andDhaA.D106C protein mutants, capable of generating covalent linkages withalkylhalide substrates, contain replacements of the catalytic triad His(H) and Asp (D) residues with Phe (F) and Cys (C), respectively.

FIG. 2C illustrates the mechanism of covalent intermediate formation byDhaA.H272F with an alkylhalide substrate. Nucleophilic displacement ofthe halide group by Asp106 is followed by the formation of the covalentester intermediate. Replacement of His272 with a Phe residue preventswater activation and traps the covalent intermediate.

FIG. 2D depicts the mechanism of covalent intermediate formation byDhaA.D106C with an alkylhalide substrate. Nucleophilic displacement ofthe halide by the Cys106 thiolate generates a thioether intermediatethat is stable to hydrolysis.

FIG. 2E depicts a structural model of the DhaA.H272F variant with acovalently attached carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl ligandsituated in the active site activity. The red shaded residues near thecap and core domain interface represent H272F and the D106 nucleophile.The yellow shaded residues denote the positions of E130 and thehalide-chelating residue W107.

FIG. 2F shows a structural model of the DhaA.H272F substrate bindingtunnel.

FIG. 3A illustrates the physical map of plasmid pGEX5X3DhaA.H272F.FLAG.This plasmid and pGEX5X3DhaA.D106C.FLAG (not shown) were used as theparental templates in mutagenesis and screening studies. The DhaA codingregions are fused at the N-terminus with glutathione S-transferase (GST)and at the C-terminus with the FLAG epitope. A Factor Xa cleavage siteis situated between the GST and DhaA coding regions.

FIG. 3B shows the purification of GST-DhaA fusion proteins. DhaA.WT (oddnumbered lanes) and DhaA.H272F (even numbered lanes) fusion proteinswere found to be soluble and efficiently purified on GSS-Sepharose 4FF(lanes 3 and 4-crude E. coli supernatant; lanes 5 and 6-washes; lanes 7through 10-purified proteins). Treatment of the fusion proteins withFactor Xa led to the formation of two proteins, GST and DhaA (WT orH272F mutant; lanes 11 and 12, respectively). Moreover, GST wasefficiently removed on GSS-Sepharose 4FF (DhaA.WT or mutant; lanes 13and 14, respectively). All proteins had the predicted molecular weight.

FIG. 4 illustrates the hydrolysis of 1-Cl-butane by DhaA.WT and mutantDhaAs.

FIGS. 5A-B show precipitation of DhaA.WT and DhaA.H272F/A/G/Q mutantswith various concentrations of (NH₄)₂SO₄. Lanes 1, 5, and 9, 0%(NH₄)₂SO₄; lanes 2, 6, and 10, 10% (NH₄)₂SO₄; lanes 3, 7, and 11, 10-45%(NH₄)₂SO₄; and lanes 4, 8, and 12, 45-70% (NH₄)₂SO₄. FIG. 5A: lanes 1-4,DhaA.WT; lanes 5-8, DhaA.H272G; and lanes 9-12, DhaA.H272Q. FIG. 5B:lanes 1-4, DhaA.WT; lanes 5-8, DhaA.H272F; and lanes 9-12, DhaA.H272A.

FIG. 6 depicts the substrate specificity of wild-type DhaA. Using aphenol red-based assay (E₅₅₈), the initial rate of the reaction wasdetermined during the first 60 seconds after enzyme addition by four 15second readings.

FIGS. 7A-B show substrates for DhaA which include a functional group(e.g., 5-(and 6-)-carboxyfluorescein, Anth (anthracene) or biotin) and alinker. “Biotin-14-Cl” refers to biotin-C₁₀H₂₁NO₂—Cl; “biotin-X-14-Cl”refers to biotin-C₁₆H₃₂N₂O₃—Cl; and “biotin-PEG4-14-Cl” refers tobiotin-C₂₁H₄₂N₂O₇—Cl.

FIG. 8A shows a HPLC separation of products ofcarboxyfluorescein-C₁₀H₂₁NO₂—Cl hydrolysis by DhaA.WT and DhaA.H272F.

FIG. 8B shows a HPLC analysis of product (as a percent of substrate)generated by DhaA.WT and DhaA.H272F hydrolysis ofcarboxyfluorescein-C₁₀H₂₁NO₂—Cl over time.

FIG. 9A-B shows SDS-PAGE analysis of the binding of DhaA.WT (lanes 1, 3,and 5 in FIG. 9A and lanes 1-8 in FIG. 9B) and DhaA.H272F (lanes 2, 4,and 6 in FIG. 9A and lanes 9-14 in FIG. 9B), tocarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (lanes 1 and 2 in FIG. 9A);carboxy-X-rhodamine-C₁₀H₂₁NO₂—Cl (lanes 3 and 4 in FIG. 9A);carboxyfluorescein-C₁₀H₂₁NO₂—Cl (lanes 5 and 6 in FIG. 9A); orbiotin-C₁₀H₂₁NO₂—Cl (B). The concentration of biotin-C₁₀H₂₁NO₂—Cl inFIG. 9B as: 0 μM (lanes 1 and 8), 125 μM (lanes 2 and 9) 25 μM (lanes 3and 10), 5 μM (lanes 4 and 11), 1 μM (lanes 5 and 12), 0.2 μM (lanes 6and 13), and 0.04 μM (lanes 7 and 14).

FIG. 10 illustrates that pretreatment of a mutant DhaA with a substrate,biotin-C₁₀H₂₁NO₂—Cl, blocks binding of another substrate. DhaA.WT-lanes1 and 2; DhaA.H272 mutants: F, lanes 3 and 4; G, lanes 5 and 6; A, lanes7 and 8; and Q, lanes 9 and 10. Samples 2, 4, 6, 8, and 10 werepretreated with biotin-C₁₀H₂₁NO₂—Cl.

FIGS. 11A-B show MALDI-TOF analysis of enzyme substrate complexes. Massspectra of DhaA.WT (A) or DhaA.H272F (B) GST fusions incubated withcarboxyfluorescein-C₁₀H₂₁NO₂—Cl.

FIG. 12 illustrates SDS-PAGE analysis of the binding properties of DhaAmutants with substitutions at residue 106, and DhaA mutants withsubstitutions at residue 106 and residue 272, tocarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl. 2 μg of protein and 25 μMcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl in 32 μl were incubated for onehour at room temperature. 10 μl of each reaction was loaded per lane.Lane 1-DhaA.D106C; lane 2-DhaA.D106C: H272F; lane 3-DhaA.D106E; lane4-DhaA.D106E:H272F; lane 5-DhaA.D106Q; lane 6-DhaA.D106Q:H272F; lane7-DhaA.WT; and lane 8-DhaA.H272F. The gel was imaged with a 570 nmfilter.

FIG. 13 depicts analysis of Renilla luciferase activity in sampleshaving a fusion of luciferase and DhaA.H272 tethered to a solid support(a streptavidin coated plate). Capture of the fusion was accomplishedusing a substrate of DhaA (i.e., biotin-C₁₀H₂₁NO₂—Cl). No activity wasfound in fractions with a fusion of Renilla luciferase and DhaA.WT.

FIG. 14A-B shows SDS-PAGE analysis of two-fold serial dilutions of E.coli expressing either DhaA.WT (lanes 1-4 of A and B) or DhaA.H272F(lanes 5-7 of A and B) treated with biotin-C₁₀H₂₁NO₂—Cl (A) orcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (B) in vivo. Arrows markproteins with M_(r) corresponding to M_(r) of DhaA.

FIG. 15 shows the binding of carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl toeukaryotic cell proteins in vivo. Two-fold serial dilutions of proteinsfrom CHO-K1 cells expressing either DhaA.WT-Flag (lanes 1-4) orDhaA.H272F-Flag (lanes 5-8) were treated withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl. Arrows mark proteins with Mrcorresponding to Mr of DhaA-Flag.

FIGS. 16A-C illustrate the permeability ofcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl to CHO-K1 cells. CHO-K1 cells(FIG. 16A, bright field image) were treated withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (25 μM, for 5 minutes at 37°C.) and quickly washed with PBS (B). FIG. 16C shows the cells after thewashing procedure.

FIG. 17A-F shows images of cells transfected withGFP-connector-DhaA.WT-Flag or GFP-connector-DhaA.H272F-Flag. CHO-K1cells were transfected with DNA coding GFP-connector-DhaA.WT-Flag (A-C)or GFP-connector-DhaA.H272F-Flag (D-F) and treated withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl. FIG. 17A, D-bright field; FIG.17B, E-GFP filter set; and FIG. 17C, F-carboxytetramethylrhodaminefilter set.

FIG. 18 shows Western blot analysis of proteins from cells transfectedwith GFP-connector-DhaA.WT-Flag (lanes 1-4) orGFP-connector-DhaA.H272F-Flag (lanes 5-8). CHO-K1 cells were transfectedwith either GFP-connector-DhaA.WT-Flag or GFP-connector-DhaA.H272F-Flagand then treated with carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (25 μM)for 0, 5, 15 or 60 minutes, washed with PBS (4×1.0 ml), and collected inSDS-sample buffer. The samples were resolved on SDS-PAGE, and analyzedon a fluoroimager. Lanes 1-4, GFP-connector-DhaA.WT-Flag treated for 0,5, 15, or 60 minutes, respectively. Lanes 5-8,GFP-connector-DhaA.H272F-Flag treated for 0, 5, 15, 60 minutes,respectively. Arrows mark proteins with M_(r) corresponding to M_(r) ofGFP-connector-DhaA.H272F-Flag.

FIGS. 19A-B illustrate the toxicity of selected substrates (FIG. 19A,carboxytetramethylrhodamine and FIG. 19B, carboxy-X-rhodamine) forCHO-K1 cells.

FIG. 20A-F illustrates a reaction scheme for a serine beta-lactamase.The reaction begins with the formation of a precovalent encountercomplex (FIG. 20A), and moves through a high-energy acylationtetrahedral intermediate (FIG. 20B) to form a transiently stableacyl-enzyme intermediate, forming an ester through the catalytic residueSer70 (FIG. 20C). Subsequently, the acyl-enzyme is attacked byhydrolytic water (FIG. 20D) to form a high-energy deacylationintermediate (FIG. 20E) (Minasov et al., 2002), which collapses to formthe hydrolyzed product (FIG. 20F). The product is then expelled,regenerating free enzyme.

FIG. 21 shows hydrolysis of FAP by GST-BlaZ over time.

FIG. 22 shows the binding of bocellin to fusions of GST and BlaZ.E166D,BlaZ.N170Q or BlaZ.E166D:N170Q. Lane 1-dye/no BlaZ; lane 2-BlaZ.WT; lane3-BlaZ.E166D; lane 4-BlaZ.N170Q; and lane 5-BlaZ.E166D:N170Q.

FIG. 23 shows the binding of CCF2 to fusions of GST and BlaZ.E166D,BlaZ.N170Q or BlaZ.E166D:N170Q. Lane 1—dye/no BlaZ; lane 2—GST-BlaZ.WT;lane 3—GST-BlaZ.E166D; lane 4—GST-BlaZ.N170Q; and lane5—GST-BlaZ.E166D:N170Q.

FIG. 24 provides fluorescence and DIC images of living CHO-K1 cellstransfected with a construct encoding GFP-connector-DhaA.H272F-NLS3 andstained with carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl.Carboxytetramethylrhodamine filter—top left; GFP filter—top right; “A”and “B” overlaid—bottom left; overlaid image “C” and DIC image of thecell—bottom right. NLS3=tandem repeat of a nuclear localization sequencefrom SV40 T antigen.

FIG. 25A-B shows fluorescence images of living CHO-K1 cells transfectedwith a construct encoding GFP-β-arrestin2 (A) and a construct encodingDhaA.H272F-β-arrestin2 and stained withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (B).

FIG. 26 shows an SDS-PAGE analysis of DhaA expression in E. coli. Lanes:1, Molecular weight standards; 2, DhaA.WT crude lysate; 3, DhaA.WTcell-free lysate; 4, DhaA.H272F crude lysate; 5, DhaA.H272F cell-freelysate; 6, vector control crude lysate; 7, vector control cell-freelysate; 8, DhaA.E130Q C1 crude lysate; 9, DhaA.E130Q C1 cell-freelysate; 10, DhaA.E130L A5 crude lysate; 11, DhaA.E130L A5 cell-freelysate; 12, DhaA.E130A A12 crude lysate; 13, DhaA.E130A A12 cell-freelysate; 14, Molecular weight standards. The arrow indicates the locationof the DhaA protein. −s, lysate before centrifugation; +s, lysate aftercentrifugation.

FIG. 27 shows an immunoblot analysis of DhaA containing lysates. Lanes:1, DhaA.WT crude lysate; 2, DhaA.WT cell-free lysate; 3, DhaA.H272Fcrude lysate; 4, DhaA.H272F cell-free lysate; 5, vector control crudelysate; 6, vector control cell-free lysate; 7, Molecular weightstandards; 8, DhaA.E130Q C1 crude lysate; 9, DhaA.E130Q C1 cell-freelysate; 10, DhaA.E130L A5 crude lysate; 11, DhaA.E130L A5 cell-freelysate; 12, DhaA.E130A A12 crude lysate; 13, DhaA.E130A A12 cell-freelysate; 14, Molecular weight standards. The arrow indicates the locationof the DhaA protein.

FIG. 28 provides fluoroimage analysis of in vitro covalent alkyl-enzymeformation. Lanes: 1, Fluorescent molecular weight standards; 2, DhaA.WT;3, DhaA.H272F mutant; 4, DhaA- (vector only control); 5, DhaA.E130Qmutant; 6, DhaA.E130L mutant; 7, DhaA.E130A mutant. The arrow indicatesthe location of the fluorescent enzyme-alkyl covalent intermediate.

FIG. 29 provides fluoroimage analysis of covalent alkyl-enzyme formationin whole cells. Lanes: 1, Fluorescent molecular weight standards; 2,DhaA.WT; 3, DhaA.H272F; 4, DhaA- (vector only control); 5, DhaA.E130Q;6, DhaA.E130L; 7, DhaA.E130A; 8, Fluorescent molecular weight standards.The arrow indicates the location of the fluorescent enzyme-alkylcovalent intermediate.

FIGS. 30A-B show Western blot analyses of DhaA-Flag captured onstreptavidin (SA) coated beads. CHO-K1 cells transiently expressingDhaA.H272F-Flag were treated with (A) or without (B) biotin-C₁₀H₂₁NO₂—Cl(25 μM, 0.1% DMSO, 60 minutes, 37° C.). Excess biotin-C₁₀H₂₁N₁O₂—Cl waswashed out, cells were lysed, and 10 μl of cell lysate was incubatedwith 5 μl of SA-coated beads (Pierce) for 60 minutes at room temperature(RT). Cell lysates (lane 1), proteins which were not bound to beads(lane 2), and proteins which were bound to beads (lane 3) were resolvedon SDS-PAGE, transferred to nitrocellulose membrane, and probed withanti-Flag antibody (Sigma).

FIGS. 30C-D illustrate analyses of hR.Luc-DhaA captured on SA coatedbeads. CHO-K1 cells transiently expressinghR.Luc-connector-DhaA.H272F-Flag were treated with or withoutbiotin-C₁₀H₂₁N₁O₂—Cl (25 μM, 0.1% DMSO, 60 minutes, 37° C.). Cells werelysed, and 10 μl of cell lysate was incubated with 5 μl of SA-coatedbeads (Pierce) for 60 minutes at room temperature. Unbound material waswashed out, and hR.Luc activity determined using Promega's “RenillaLuciferase Assay System” (C) or captured hR.Luc analyzed by Western blot(D). C) Column 1, cells treated with biotin-C₁₀H₂₁NO₂—Cl, and excessbiotin-C₁₀H₂₁NO₂—Cl washed out; column 2, untreated cells; and column 3,cells treated with biotin-C₁₀H₂₁NO₂—Cl without washing out excessbiotin-C₁₀H₂₁N₁O₂—Cl. D) Cell lysate (lane 1), proteins which were notbound to beads (lane 2), and proteins which were bound to beads (lane 3)were resolved on SDS-PAGE, transferred to nitrocellulose membrane, andprobed with anti-R.Luc antibody (Chemicon).

FIGS. 31A-B show the identification of potential improvements from aDhaA.H272F K175/C176 library using an in vivocarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl labeling screening assay (A)and the identification of potential improvements from a DhaA.H272FK175/C176 library using an anti-FLAG immobilized protein assay (B). DhaAmutants with 2-fold higher activity than the H272F parent (horizontalline) are identified by arrows in FIG. 31A. DhaA mutants with signals 3-to 4-fold higher than DhaA.H272F are identified in FIG. 31B. DhaA.H272Fparental and DhaA-controls (in triplicate) are located in wells 12C-Eand 12F-H, respectively.

FIG. 32 depicts an overview of the MagneGST™ assay developed forhigh-throughput screening of DhaA libraries usingcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl.

FIGS. 33A-B shows the identification of potential improvements (i.e.,hits) from mutant DhaA protein libraries. Representative screeningplates from the DhaA.H272F C176 (A) and DhaA.D106C K175/C176 NNK (B)libraries are shown. Arrows indicate potentially improved clones.

FIGS. 34A-B show the identification of potential improvements (i.e.,hits) from mutant DhaA protein libraries. Shown are two representativeplates from the DhaA.H272F Y273 (NNK) library screening using theMagneGST™-based screening assay. Arrows indicate potentially improvedclones.

FIGS. 35A-B show the sequence of hits at positions 175, 176 and 273 forDhaA.H272F (A) and the sequence hits at positions 175 and 176 forDhaA.D106C (B).

FIGS. 36A-B illustrate the relative activity of identified DhaA hitscompared to parental proteins in secondary assays. A). The indicatedDhaA mutants were re-assayed using the MagneGST™ assay andcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (n=3). B). The indicated DhaAmutants were analyzed using the protein immobilization assay andbiotin-PEG4-14-Cl.

FIGS. 37A-C demonstrate the relative labeling rates of purified DhaAmutants with carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl. A). SDS-PAGE andfluorimage gel analysis of labeling time-course. Lanes 1 and 2,DhaA.D106 30H4; lanes: 3 and 4, H272F; lanes 5 and 6, DhaA.H272F ES;lanes 7 and 8, DhaA.H272F H11; lanes 9 and 10, DhaA.H272F A7; lanes 11and 12, DhaA.H272F A7; lanes 13 and 14, DhaA.H272F A7YM; lanes 15 and16, DhaA.H272F YL; lanes 17 and 18, DhaA.H272F 2G7; lanes 19 and 20,DhaA.H272F 3A7; lanes 21 and 22, DhaA.H272F H11YL. Reactions in odd andeven numbered lanes were incubated for 2 and 30 minutes, respectively,at room temperature. B). SDS-PAGE and fluorimage gel analysis oflabeling time-course of first generation, DhaA.H272F A7, andsecond-generation, DhaA.H272F H11YL and DhaA.H272F A7YM mutants. Lane:1, 20 seconds; lane 2, 40 seconds; lane 3, 1 minute; lane 4, 2 minutes;and lane 5, 7 minutes. Arrows indicate the presence of fluorescentlylabeled DhaA fusion proteins. C). Rates of DhaA labeling withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl. The fluorescent products shownin FIG. 37B were quantitated and plotted versus time.

FIGS. 38A-B depict the labeling time-course of DhaA.H272F H11YL. A).SDS-PAGE and fluorimage gel analysis of purified DhaA.H272F H11YL withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl. The indicated times are inseconds. An arrow indicates the location of labeled DhaA.H272F H11YL.B). Rate of DhaA.H272F H11YL labeling. The fluorescent products shown inFIG. 38A were quantitated and plotted versus time.

FIGS. 39A-B show fluorescence polarization analysis of DhaA mutantsusing (A) carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl and (B)carboxyfluorescein-C₁₀H₂₁NO₂—Cl.

FIG. 40 shows the second order rate constants (M⁻¹ sec⁻¹) of parental(DhaA.H272F and DhaA.D106C), and first and second generation DhaAmutants determined by fluorescence polarization (FP).

FIG. 41 illustrates a comparison of the labeling rates of DhaA.H272FH11YL (“HaloTag”) to carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl andcarboxytetramethylrhodamine-coupled biotin to streptavidin usingfluorescence polarization.

FIG. 42A-D depicts the structural models of the DhaA.H272F andDhaA.H272F H11YL substrate tunnels without (FIGS. 42A and C) and with(FIGS. 42B and D) carboxytetramethylrhodamine-coupled substrate.

FIG. 43A-B shows the results of thermostability studies of purified DhaAproteins. A). Analysis of DhaA.H272F parental and select firstgeneration DhaA mutants. B). Analysis of DhaA.H272F-based secondgeneration DhaA mutants.

FIG. 44 demonstrates the effect of low temperature on DhaA.H272F H11YLreaction rates. Following incubation at either 4° C. or 23° C., 10 μLaliquots of the reaction mixture were quickly added to an equivalentamount of SDS-loading dye preincubated at 95° C. The resulting SDS-PAGEgel was examined by fluorimage analysis.

FIG. 45A-B illustrates the immobilization of DhaA to solid supports. A).General reaction scheme between DhaA.H272F mutants and immobilizedbiotin-chloroalkanes. B). Titration of select DhaA mutants againstimmobilized biotin-PEG-14-Cl.

FIGS. 46A-B show the in vivo labeling of parental and first generationDhaA mutants expressed in CHO-K1 cells. A). SDS-PAGE fluorimage gelanalysis of DhaA mutants following in vivo labeling withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (5 μM). Lanes: 1-4, DhaA.H272F;lanes 5-8, DhaA.H272F A7; lanes 9-12, DhaA.H272F H11; and lanes 13-16,DhaA.D106C. Each lane in the series represents 5, 15, 30 and 120 minutetime points. An arrow denotes the location of labeled DhaA. B).Quantitation of carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl binding to DhaAmutants.

FIGS. 46C-D demonstrate the in vivo labeling of first and secondgeneration DhaA mutants expressed in CHO-K1 cells. C). SDS-PAGEfluorimage gel analysis of DhaA mutants following in vivo labeling withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (5 μM). Lanes: 1-4, DhaA.H272FA7; lanes 5-8, DhaA.H272F H11YL; and lanes 9-12, DhaA.D106C 30H4. Eachlane in the series represents 5, 15, 30 and 120 minute time points. D).Quantitation of carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl binding to DhaAmutants.

FIG. 47A-C show the labeling of DhaA.H272F A7 and DhaA.H272F H11YL withdifferent concentrations of carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl inmammalian cell lysates.

FIGS. 48A-B show the stability of parental and first generation DhaAmutants in vivo. A). Fluorimage gel analysis ofcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl labeled DhaA mutants. Lanes:1-3, DhaA.H272F; lanes 4-6, DhaA.D106C; lanes 7-9, DhaA.H272F A7; lanes10-12, DhaA.H272F H11; and lane 13 standards. Lanes 1, 4, 7 and 10represent samples taken 12 hours post-transfection. Lanes 2, 5, 8, and11 represent samples taken 24 hours post-transfection. Lanes 3, 5, 8 and12 represent samples taken 48 hours post-transfection. Arrow indicatesthe location of labeled DhaA mutants. B). Quantitation of fluorimagedgel.

FIGS. 48C-D shows a comparison of the stability of DhaA.H272 mutants invivo. A). Fluorimage gel analysis ofcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl labeled DhaA mutants. Lanes:1-3, DhaA.H272F A7; lanes 4-6, DhaA.H272F H11YL; and lane 7 standards.Lanes 1 and 4 represent samples taken 12 hours post-transfection. Lanes2 and 5 represent samples taken 24 hours post-transfection. Lanes 3 and6 represent samples taken 48 hours post-transfection. Arrow indicateslocation of DhaA variants. B). Quantitation of the fluorimaged gel.

FIG. 49 shows the nucleotide (SEQ ID NO:80) and amino acid (SEQ IDNO:81) sequence of DhaA.H272 H11YL which is in pHT2. The restrictionsites listed were incorporated to facilitate generation of functional N-and C-terminal fusions.

FIGS. 50A-B provide fluorescence and DIC images of living HeLa cellstransfected with a vector coding for DhaA.H272F H11YL stained withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl, and counterstained withMitoTracker® Green FM (left panel); or stained withDiAc-carboxyfluorescein-C₁₀H₂₁NO₂—Cl and counterstained withMitoTracker® Orange CMTMRos (right panel). Carboxyfluorescein filter-topleft; carboxytetramethylrhodamine filter-top right; carboxyfluoresceinand carboxytetramethylrhodamine overlaid image-bottom right; DIC imageof the cell-bottom left.

FIGS. 50C-D provide fluorescence and DIC images of living CHO-K1 cellstransfected with a vector plasmid pHT2 coding for DhaA.H272F H11YL HT2(see FIG. 49) or pCIneo harboring DhaA.H272F, stained with 0.2, 1.0 or5.0 μM carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl, and fixed with 3.7% ofparaformaldehyde. Carboxytetramethylrhodamine filter-left image of eachpanel; carboxytetramethylrhodamine and DIC overlaid image-right image ineach panel.

FIGS. 50E-F depict the localization of β-arrestin2-DhaA.H272F H11YLHT2protein fusions in HeLa cells. Photomicrographs ofDiAc-carboxyfluorescein-C₁₀H₂₁NO₂—Cl (E) andcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (F) labeled cells.

FIG. 51A-B show the capture of a hRLuc-DhaA.H272F H11YLHT2 fusionprotein expressed in transiently transfected CHO-K1 cells. CapturinghRLuc activity on streptavidin coated 96-well plates (A) andstreptavidin-MagneSphere paramagnetic particles (B).

FIGS. 52A-C show the relative labeling rates of and product accumulationfor purified DhaA.H272F H11YL withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl,carboxytetramethylrhodamine-p-phenethyl-Cl andcarboxytetramethylrhodamine-furanyl-propyl-Cl. A). Fluorescencepolarization (FP) analysis. B). SDS-PAGE and fluorimage gel analysis.C). Quantitation of fluorescent product accumulation.

FIGS. 53A-C show the relative labeling rates of and product accumulationfor purified DhaA.H272F H11YL with carboxyfluorescein-C₁₀H₂₁NO₂—Cl,carboxyfluorescein-p-phenethyl-Cl andcarboxyfluorescein-furanyl-propyl-Cl. A). Fluorescence polarization (FP)analysis. B). SDS-PAGE and fluorimage gel analysis. C). Quantitation offluorescent product accumulation.

FIGS. 54A-B demonstrate the in vivo labeling of DhaA.H272F H11YL withdifferent carboxytetramethylrhodamine chloroalkanes. A). Fluorimage gelanalysis. Lanes: 1-3, carboxytetramethylrhodamine-14-Cl, 5, 15 and 60minutes, respectively; lanes 4-6,carboxytetramethylrhodamine-furanyl-propyl-Cl, 5, 15 and 60 minutes,respectively; and lanes 7-9, carboxytetramethylrhodamine-p-phenethyl-Cl,5, 15 and 60 minutes, respectively. B). Quantitation of theDhaA.H11Y273L in vivo labeling rates using 1, 5 and 20 μM substrate.

FIGS. 55A-B demonstrate the reactivity of immobilized DhaA.H272F H11YLto biotin-coupled chloroalkane substrates. A). General reaction anddetection scheme. B). Reactivity of biotin-PEG4-14-Cl, biotin-14-Cl andbiotin-p-phenethyl-14-Cl with immobilized DhaA.H272F H11Y273L protein.

FIG. 56 shows exemplary substrates with ring structures.

FIG. 57 illustrates the use of a hydrolase substrate of the inventionand a mutant hydrolase of the invention for the immobilization andcapture of proteins of interest.

FIG. 58 is a schematic of a hydrolase substrate of the invention and amutant hydrolase of the invention for immunoprecipitation.

FIGS. 59A-B illustrate the use of a hydrolase substrate of the inventionand a mutant hydrolase of the invention to detect cAMP.

FIG. 60A is a schematic of a cell-based protease detection system.

FIG. 60B is a schematic of a cell-based protease detection system.

FIG. 60C is a schematic of the use of short lived reporters to detect aprotease of interest.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

A “nucleophile” is a molecule which donates electrons.

A “selectable marker protein” encodes an enzymatic activity that confersto a cell the ability to grow in medium lacking what would otherwise bean essential nutrient (e.g., the TRP1 gene in yeast cells) or in amedium with an antibiotic or other drug, i.e., the expression of thegene encoding the selectable marker protein in a cell confers resistanceto an antibiotic or drug to that cell relative to a corresponding cellwithout the gene. When a host cell must express a selectable marker togrow in selective medium, the marker is said to be a positive selectablemarker (e.g., antibiotic resistance genes which confer the ability togrow in the presence of the appropriate antibiotic). Selectable markerscan also be used to select against host cells containing a particulargene (e.g., the sacB gene which, if expressed, kills the bacterial hostcells grown in medium containing 5% sucrose); selectable markers used inthis manner are referred to as negative selectable markers orcounter-selectable markers. Common selectable marker gene sequencesinclude those for resistance to antibiotics such as ampicillin,tetracycline, kanamycin, puromycin, bleomycin, streptomycin, hygromycin,neomycin, Zeocin™, and the like. Selectable auxotrophic gene sequencesinclude, for example, hisD, which allows growth in histidine free mediain the presence of histidinol. Suitable selectable marker genes includea bleomycin-resistance gene, a metallothionein gene, a hygromycinB-phosphotransferase gene, the AURI gene, an adenosine deaminase gene,an aminoglycoside phosphotransferase gene, a dihydrofolate reductasegene, a thymidine kinase gene, a xanthine-guaninephosphoribosyltransferase gene, and the like.

A “nucleic acid”, as used herein, is a covalently linked sequence ofnucleotides in which the 3′ position of the pentose of one nucleotide isjoined by a phosphodiester group to the 5′ position of the pentose ofthe next, and in which the nucleotide residues (bases) are linked inspecific sequence, i.e., a linear order of nucleotides, and includesanalogs thereof, such as those having one or more modified bases, sugarsand/or phosphate backbones. A “polynucleotide”, as used herein, is anucleic acid containing a sequence that is greater than about 100nucleotides in length. An “oligonucleotide” or “primer”, as used herein,is a short polynucleotide or a portion of a polynucleotide. The term“oligonucleotide” or “oligo” as used herein is defined as a moleculecomprised of 2 or more deoxyribonucleotides or ribonucleotides,preferably more than 3, and usually more than 10, but less than 250,preferably less than 200, deoxyribonucleotides or ribonucleotides. Theoligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, amplification, e.g., polymerase chainreaction (PCR), reverse transcription (RT), or a combination thereof. A“primer” is an oligonucleotide which is capable of acting as a point ofinitiation for nucleic acid synthesis when placed under conditions inwhich primer extension is initiated. A primer is selected to have on its3′ end a region that is substantially complementary to a specificsequence of the target (template). A primer must be sufficientlycomplementary to hybridize with a target for primer elongation to occur.A primer sequence need not reflect the exact sequence of the target. Forexample, a non-complementary nucleotide fragment may be attached to the5′ end of the primer, with the remainder of the primer sequence beingsubstantially complementary to the target. Non-complementary bases orlonger sequences can be interspersed into the primer provided that theprimer sequence has sufficient complementarity with the sequence of thetarget to hybridize and thereby form a complex for synthesis of theextension product of the primer. Primers matching or complementary to agene sequence may be used in amplification reactions, RT-PCR and thelike.

Nucleic acid molecules are said to have a “5′-terminus” (5′ end) and a“3′-terminus” (3′ end) because nucleic acid phosphodiester linkagesoccur to the 5′ carbon and 3′ carbon of the pentose ring of thesubstituent mononucleotides. The end of a polynucleotide at which a newlinkage would be to a 5′ carbon is its 5′ terminal nucleotide. The endof a polynucleotide at which a new linkage would be to a 3′ carbon isits 3′ terminal nucleotide. A terminal nucleotide, as used herein, isthe nucleotide at the end position of the 3′- or 5′-terminus.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides referred to as the “5′end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring.

As used herein, a nucleic acid sequence, even if internal to a largeroligonucleotide or polynucleotide, also may be said to have 5′ and 3′ends. In either a linear or circular DNA molecule, discrete elements arereferred to as being “upstream” or 5′ of the “downstream” or 3′elements. This terminology reflects the fact that transcription proceedsin a 5′ to 3′ fashion along the DNA strand. Typically, promoter andenhancer elements that direct transcription of a linked gene (e.g., openreading frame or coding region) are generally located 5′ or upstream ofthe coding region. However, enhancer elements can exert their effecteven when located 3′ of the promoter element and the coding region.Transcription termination and polyadenylation signals are located 3′ ordownstream of the coding region.

The term “codon” as used herein, is a basic genetic coding unit,consisting of a sequence of three nucleotides that specify a particularamino acid to be incorporation into a polypeptide chain, or a start orstop signal. The term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. Typically, the coding region is bounded on the 5′ side bythe nucleotide triplet “ATG” which encodes the initiator methionine andon the 3′ side by a stop codon (e.g., TAA, TAG, TGA). In some cases thecoding region is also known to initiate by a nucleotide triplet “TTG”.

As used herein, the terms “isolated and/or purified” refer to in vitropreparation, isolation and/or purification of a nucleic acid molecule, apolypeptide, peptide or protein, so that it is not associated with invivo substances. Thus, the term “isolated” when used in relation to anucleic acid, as in “isolated oligonucleotide” or “isolatedpolynucleotide” refers to a nucleic acid sequence that is identified andseparated from at least one contaminant with which it is ordinarilyassociated in its source. An isolated nucleic acid is present in a formor setting that is different from that in which it is found in nature.In contrast, non-isolated nucleic acids (e.g., DNA and RNA) are found inthe state they exist in nature. For example, a given DNA sequence (e.g.,a gene) is found on the host cell chromosome in proximity to neighboringgenes; RNA sequences (e.g., a specific mRNA sequence encoding a specificprotein), are found in the cell as a mixture with numerous other mRNAsthat encode a multitude of proteins. Hence, with respect to an “isolatednucleic acid molecule”, which includes a polynucleotide of genomic,cDNA, or synthetic origin or some combination thereof, the “isolatednucleic acid molecule” (1) is not associated with all or a portion of apolynucleotide in which the “isolated nucleic acid molecule” is found innature, (2) is operably linked to a polynucleotide which it is notlinked to in nature, or (3) does not occur in nature as part of a largersequence. The isolated nucleic acid molecule may be present insingle-stranded or double-stranded form. When a nucleic acid molecule isto be utilized to express a protein, the nucleic acid contains at aminimum, the sense or coding strand (i.e., the nucleic acid may besingle-stranded), but may contain both the sense and anti-sense strands(i.e., the nucleic acid may be double-stranded).

The term “wild-type” as used herein, refers to a gene or gene productthat has the characteristics of that gene or gene product isolated froma naturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “wild-type” form of the gene. In contrast, the term “mutant” refersto a gene or gene product that displays modifications in sequence and/orfunctional properties (i.e., altered characteristics) when compared tothe wild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct.

The term “recombinant DNA molecule” means a hybrid DNA sequencecomprising at least two nucleotide sequences not normally found togetherin nature. The term “vector” is used in reference to nucleic acidmolecules into which fragments of DNA may be inserted or cloned and canbe used to transfer DNA segment(s) into a cell and capable ofreplication in a cell. Vectors may be derived from plasmids,bacteriophages, viruses, cosmids, and the like.

The terms “recombinant vector”, “expression vector” or “construct” asused herein refer to DNA or RNA sequences containing a desired codingsequence and appropriate DNA or RNA sequences necessary for theexpression of the operably linked coding sequence in a particular hostorganism. Prokaryotic expression vectors include a promoter, a ribosomebinding site, an origin of replication for autonomous replication in ahost cell and possibly other sequences, e.g. an optional operatorsequence, optional restriction enzyme sites. A promoter is defined as aDNA sequence that directs RNA polymerase to bind to DNA and to initiateRNA synthesis. Eukaryotic expression vectors include a promoter,optionally a polyadenylation signal and optionally an enhancer sequence.

A polynucleotide having a nucleotide sequence “encoding a peptide,protein or polypeptide” means a nucleic acid sequence comprising thecoding region of a gene, or a fragment thereof which encodes a geneproduct having substantially the same activity as the correspondingfull-length peptide, protein or polypeptide. The coding region may bepresent in either a cDNA, genomic DNA or RNA form. When present in a DNAform, the oligonucleotide may be single-stranded (i.e., the sensestrand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if neededto permit proper initiation of transcription and/or correct processingof the primary RNA transcript. Alternatively, the coding region utilizedin the expression vectors of the present invention may containendogenous enhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. In further embodiments, the coding regionmay contain a combination of both endogenous and exogenous controlelements.

The term “transcription regulatory element” or “transcription regulatorysequence” refers to a genetic element or sequence that controls someaspect of the expression of nucleic acid sequence(s). For example, apromoter is a regulatory element that facilitates the initiation oftranscription of an operably linked coding region. Other regulatoryelements include, but are not limited to, transcription factor bindingsites, splicing signals, polyadenylation signals, termination signalsand enhancer elements, and include elements which increase or decreasetranscription of linked sequences, e.g., in the presence of trans-actingelements.

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription. Promoter and enhancer elements have been isolated froma variety of eukaryotic sources including genes in yeast, insect andmammalian cells. Promoter and enhancer elements have also been isolatedfrom viruses and analogous control elements, such as promoters, are alsofound in prokaryotes. The selection of a particular promoter andenhancer depends on the cell type used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types. Forexample, the SV40 early gene enhancer is very active in a wide varietyof cell types from many mammalian species and has been widely used forthe expression of proteins in mammalian cells. Two other examples ofpromoter/enhancer elements active in a broad range of mammalian celltypes are those from the human elongation factor 1 gene and the longterminal repeats of the Rous sarcoma virus; and the humancytomegalovirus.

The term “promoter/enhancer” denotes a segment of DNA containingsequences capable of providing both promoter and enhancer functions(i.e., the functions provided by a promoter element and an enhancerelement as described above). For example, the long terminal repeats ofretroviruses contain both promoter and enhancer functions. Theenhancer/promoter may be “endogenous” or “exogenous” or “heterologous.”An “endogenous” enhancer/promoter is one that is naturally linked with agiven gene in the genome. An “exogenous” or “heterologous”enhancer/promoter is one that is placed in juxtaposition to a gene bymeans of genetic manipulation (i.e., molecular biological techniques)such that transcription of the gene is directed by the linkedenhancer/promoter.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript ineukaryotic host cells. Splicing signals mediate the removal of intronsfrom the primary RNA transcript and consist of a splice donor andacceptor site (Sambrook et al., 1989). A commonly used splice donor andacceptor site is the splice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly(A) site” or“poly(A) sequence” as used herein denotes a DNA sequence which directsboth the termination and polyadenylation of the nascent RNA transcript.Efficient polyadenylation of the recombinant transcript is desirable, astranscripts lacking a poly(A) tail are unstable and are rapidlydegraded. The poly(A) signal utilized in an expression vector may be“heterologous” or “endogenous.” An endogenous poly(A) signal is one thatis found naturally at the 3′ end of the coding region of a given gene inthe genome. A heterologous poly(A) signal is one which has been isolatedfrom one gene and positioned 3′ to another gene. A commonly usedheterologous poly(A) signal is the SV40 poly(A) signal. The SV40 poly(A)signal is contained on a 237 bp BamH I/Bcl I restriction fragment anddirects both termination and polyadenylation (Sambrook et al., 1989).

Eukaryotic expression vectors may also contain “viral replicons” or“viral origins of replication.” Viral replicons are viral DNA sequenceswhich allow for the extrachromosomal replication of a vector in a hostcell expressing the appropriate replication factors. Vectors containingeither the SV40 or polyoma virus origin of replication replicate to highcopy number (up to 10⁴ copies/cell) in cells that express theappropriate viral T antigen. In contrast, vectors containing thereplicons from bovine papillomavirus or Epstein-Barr virus replicateextrachromosomally at low copy number (about 100 copies/cell).

The term “in vitro” refers to an artificial environment and to processesor reactions that occur within an artificial environment. In vitroenvironments include, but are not limited to, test tubes and celllysates. The term “in situ” refers to cell culture. The term “in vivo”refers to the natural environment (e.g., an animal or a cell) and toprocesses or reaction that occur within a natural environment.

The term “expression system” refers to any assay or system fordetermining (e.g., detecting) the expression of a gene of interest.Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used. A wide range ofsuitable mammalian cells are available from a wide range of sources(e.g., the American Type Culture Collection, Rockland, Md.). The methodof transformation or transfection and the choice of expression vehiclewill depend on the host system selected. Transformation and transfectionmethods are described, e.g., in Sambrook et al., 1989. Expressionsystems include in vitro gene expression assays where a gene of interest(e.g., a reporter gene) is linked to a regulatory sequence and theexpression of the gene is monitored following treatment with an agentthat inhibits or induces expression of the gene. Detection of geneexpression can be through any suitable means including, but not limitedto, detection of expressed mRNA or protein (e.g., a detectable productof a reporter gene) or through a detectable change in the phenotype of acell expressing the gene of interest. Expression systems may alsocomprise assays where a cleavage event or other nucleic acid or cellularchange is detected.

The term “gene” refers to a DNA sequence that comprises coding sequencesand optionally control sequences necessary for the production of apolypeptide from the DNA sequence. The polypeptide can be encoded by afull-length coding sequence or by any portion of the coding sequence solong as the portion encodes a gene product with substantially the sameactivity as the full-length polypeptide.

Nucleic acids are known to contain different types of mutations. A“point” mutation refers to an alteration in the sequence of a nucleotideat a single base position from the wild-type sequence. Mutations mayalso refer to insertion or deletion of one or more bases, so that thenucleic acid sequence differs from a reference, e.g., a wild-type,sequence.

As used herein, the terms “hybridize” and “hybridization” refer to theannealing of a complementary sequence to the target nucleic acid, i.e.,the ability of two polymers of nucleic acid (polynucleotides) containingcomplementary sequences to anneal through base pairing. The terms“annealed” and “hybridized” are used interchangeably throughout, and areintended to encompass any specific and reproducible interaction betweena complementary sequence and a target nucleic acid, including binding ofregions having only partial complementarity. Certain bases not commonlyfound in natural nucleic acids may be included in the nucleic acids ofthe present invention and include, for example, inosine and7-deazaguanine Those skilled in the art of nucleic acid technology candetermine duplex stability empirically considering a number of variablesincluding, for example, the length of the complementary sequence, basecomposition and sequence of the oligonucleotide, ionic strength andincidence of mismatched base pairs. The stability of a nucleic acidduplex is measured by the melting temperature, or “T_(m)”. The T_(m) ofa particular nucleic acid duplex under specified conditions is thetemperature at which on average half of the base pairs havedisassociated.

The term “stringency” is used in reference to the conditions oftemperature, ionic strength, and the presence of other compounds, underwhich nucleic acid hybridizations are conducted. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences. Thus, conditions of “medium” or “low” stringency are oftenrequired when it is desired that nucleic acids which are not completelycomplementary to one another be hybridized or annealed together. The artknows well that numerous equivalent conditions can be employed tocomprise medium or low stringency conditions. The choice ofhybridization conditions is generally evident to one skilled in the artand is usually guided by the purpose of the hybridization, the type ofhybridization (DNA-DNA or DNA-RNA), and the level of desired relatednessbetween the sequences (e.g., Sambrook et al., 1989; Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington D.C., 1985,for a general discussion of the methods).

The stability of nucleic acid duplexes is known to decrease with anincreased number of mismatched bases, and further to be decreased to agreater or lesser degree depending on the relative positions ofmismatches in the hybrid duplexes. Thus, the stringency of hybridizationcan be used to maximize or minimize stability of such duplexes.Hybridization stringency can be altered by: adjusting the temperature ofhybridization; adjusting the percentage of helix destabilizing agents,such as formamide, in the hybridization mix; and adjusting thetemperature and/or salt concentration of the wash solutions. For filterhybridizations, the final stringency of hybridizations often isdetermined by the salt concentration and/or temperature used for thepost-hybridization washes.

“High stringency conditions” when used in reference to nucleic acidhybridization include conditions equivalent to binding or hybridizationat 42EC in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42EC when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization include conditions equivalent to binding or hybridizationat 42EC in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42EC when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” include conditions equivalent to binding orhybridization at 42EC in a solution consisting of 5×SSPE (43.8 g/l NaCl,6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH),0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500 ml: 5 gFicoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 g/mldenatured salmon sperm DNA followed by washing in a solution comprising5×SSPE, 0.1% SDS at 42EC when a probe of about 500 nucleotides in lengthis employed.

By “peptide”, “protein” and “polypeptide” is meant any chain of aminoacids, regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation). Unless otherwise specified, the termsare interchangeable. The nucleic acid molecules of the invention encodea variant (mutant) of a naturally-occurring (wild-type) protein orfragment thereof which has substantially the same activity as the fulllength mutant protein. Preferably, such a mutant protein has an aminoacid sequence that is at least 85%, preferably 90%, and most preferably95% or 99%, identical to the amino acid sequence of a correspondingwild-type protein.

Polypeptide molecules are said to have an “amino terminus” (N-terminus)and a “carboxy terminus” (C-terminus) because peptide linkages occurbetween the backbone amino group of a first amino acid residue and thebackbone carboxyl group of a second amino acid residue. The terms“N-terminal” and “C-terminal” in reference to polypeptide sequencesrefer to regions of polypeptides including portions of the N-terminaland C-terminal regions of the polypeptide, respectively. A sequence thatincludes a portion of the N-terminal region of polypeptide includesamino acids predominantly from the N-terminal half of the polypeptidechain, but is not limited to such sequences. For example, an N-terminalsequence may include an interior portion of the polypeptide sequenceincluding bases from both the N-terminal and C-terminal halves of thepolypeptide. The same applies to C-terminal regions. N-terminal andC-terminal regions may, but need not, include the amino acid definingthe ultimate N-terminus and C-terminus of the polypeptide, respectively.

The term “isolated” when used in relation to a polypeptide, as in“isolated protein” or “isolated polypeptide” refers to a polypeptidethat is identified and separated from at least one contaminant withwhich it is ordinarily associated in its source. Thus, an isolatedpolypeptide (1) is not associated with proteins found in nature, (2) isfree of other proteins from the same source, e.g., free of humanproteins, (3) is expressed by a cell from a different species, or (4)does not occur in nature. In contrast, non-isolated polypeptides (e.g.,proteins and enzymes) are found in the state they exist in nature. Theterms “isolated polypeptide”, “isolated peptide” or “isolated protein”include a polypeptide, peptide or protein encoded by cDNA or recombinantRNA including one of synthetic origin, or some combination thereof.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule expressed from a recombinant DNAmolecule. In contrast, the term “native protein” is used herein toindicate a protein isolated from a naturally occurring (i.e., anonrecombinant) source. Molecular biological techniques may be used toproduce a recombinant form of a protein with identical properties ascompared to the native form of the protein.

The term “fusion polypeptide” as used herein refers to a chimericprotein containing a protein of interest (e.g., luciferase, an affinitytag or a targeting sequence) joined to a different protein, e.g., amutant hydrolase.

As used herein, the term “antibody” refers to a protein having one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as the myriad of immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively.

The basic immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies may exist as intact immunoglobulins, or as modifications in avariety of forms including, for example, FabFc₂, Fab, Fv, Fd, (FabN)₂,an Fv fragment containing only the light and heavy chain variableregions, a Fab or (Fab)N₂ fragment containing the variable regions andparts of the constant regions, a single-chain antibody, e.g., scFv,CDR-grafted antibodies and the like. The heavy and light chain of a Fvmay be derived from the same antibody or different antibodies therebyproducing a chimeric Fv region. The antibody may be of animal(especially mouse or rat) or human origin or may be chimeric orhumanized. As used herein the term “antibody” includes these variousforms.

The terms “cell,” “cell line,” “host cell,” as used herein, are usedinterchangeably, and all such designations include progeny or potentialprogeny of these designations. By “transformed cell” is meant a cellinto which (or into an ancestor of which) has been introduced a nucleicacid molecule of the invention. Optionally, a nucleic acid molecule ofthe invention may be introduced into a suitable cell line so as tocreate a stably transfected cell line capable of producing the proteinor polypeptide encoded by the nucleic acid molecule. Vectors, cells, andmethods for constructing such cell lines are well known in the art. Thewords “transformants” or “transformed cells” include the primarytransformed cells derived from the originally transformed cell withoutregard to the number of transfers. All progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.Nonetheless, mutant progeny that have the same functionality as screenedfor in the originally transformed cell are included in the definition oftransformants.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). Homology isoften measured using sequence analysis software (e.g., Sequence AnalysisSoftware Package of the Genetics Computer Group. University of WisconsinBiotechnology Center. 1710 University Avenue. Madison, Wis. 53705). Suchsoftware matches similar sequences by assigning degrees of homology tovarious substitutions, deletions, insertions, and other modifications.Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine.

The term “purified” or “to purify” means the result of any process thatremoves some of a contaminant from the component of interest, such as aprotein or nucleic acid. The percent of a purified component is therebyincreased in the sample.

The term “operably linked” as used herein refer to the linkage ofnucleic acid sequences in such a manner that a nucleic acid moleculecapable of directing the transcription of a given gene and/or thesynthesis of a desired protein molecule is produced. The term alsorefers to the linkage of sequences encoding amino acids in such a mannerthat a functional (e.g., enzymatically active, capable of binding to abinding partner, capable of inhibiting, etc.) protein or polypeptide, ora precursor thereof, e.g., the pre- or prepro-form of the protein orpolypeptide, is produced.

All amino acid residues identified herein are in the naturalL-configuration. In keeping with standard polypeptide nomenclature,abbreviations for amino acid residues are as shown in the followingTable of Correspondence.

TABLE OF CORRESPONDENCE 1-Letter 3-Letter AMINO ACID Y Tyr L-tyrosine GGly L-glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine SSer L-serine I Ile L-isoleucine L Leu L-leucine T Thr L-threonine V ValL-valine P Pro L-proline K Lys L-lysine H His L-histidine Q GlnL-glutamine E Glu L-glutamic acid W Trp L-tryptophan R Arg L-arginine DAsp L-aspartic acid N Asn L-asparagine C Cys L-cysteine

As used herein, the term “poly-histidine tract” or (His tag) refers to amolecule comprising two to ten histidine residues, e.g., apoly-histidine tract of five to ten residues. A poly-histidine tractallows the affinity purification of a covalently linked molecule on animmobilized metal, e.g., nickel, zinc, cobalt or copper, chelate columnor through an interaction with another molecule (e.g., an antibodyreactive with the His tag).

As used herein, “pure” means an object species is the predominantspecies present (i.e., on a molar basis it is more abundant than anyother individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a “substantially pure”composition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, about 90%, about 95%, and about 99%. Most preferably,the object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) wherein the composition consists essentially of a singlemacromolecular species.

A “quantum dot” is an ultrasmall, bright, highly photostablesemiconductor crystallite with a broad excitation band that a narrowemission band, i.e., it is a fluorescent crystalline nanoparticle.

As used herein, an “upconverting nanoparticle” means a nanoparticlewhich is a combination of an absorber which is excited by infrared (IR)light and an emitter ion in a crystal lattice, which converts IR lightinto visible radiation.

As used herein, a “triplet sensitizer” is a molecule or a group that issubstantially chemically inert and that can absorb light at wavelengthsthat are not or are only weakly absorbed by a substrate. The“sensitizer” can then release energy which will cause an oxygen atom inthe substrate or compound to be excited to a singlet oxygen state. Thesubstrate with an oxygen atom in a singlet oxygen state can destroymolecules, such as proteins in close proximity thereto. Examples oftriplet sensitizers include, for example, eosin or malachite green.

A radionuclide useful in a diagnostic application includes, e.g.,metallic radionuclides (i.e., metallic radioisotopes or metallicparamagnetic ions), including Antimony-124, Antimony-125, Arsenic-74,Barium-103, Barium-140, Beryllium-7, Bismuth-206, Bismuth-207,Cadmium-109, Cadmium-115m, Calcium-45, Cerium-139, Cerium-141,Cerium-144, Cesium-137, Chromium-51, Cobalt-55, Cobalt-56, Cobalt-57,Cobalt-58, Cobalt-60, Cobalt-64, Copper-67, Erbium-169, Europium-152,Gallium-64, Gallium-68, Gadolinium-153, Gadolinium-157 Gold-195,Gold-199, Hafnium-175, Hafnium-175-181, Holmium-166, Indium-110,Indium-111, Iridium-192, Iron-55, Iron-59, Krypton-85, Lead-210,Manganese-54, Mercury-197, Mercury-203, Molybdenum-99, Neodymium-147,Neptunium-237, Nickel-63, Niobium-95, Osmium-185+191, Palladium-103,Platinum-195m, Praseodymium-143, Promethium-147, Protactinium-233,Radium-226, Rhenium-186, Rhenium-188, Rubidium-86, Ruthenium-103,Ruthenium-106, Scandium-44, Scandium-46, Selenium-75, Silver-110m,Silver-111, Sodium-22, Strontium-85, Strontium-89, Strontium-90,Sulfur-35, Tantalum-182, Technetium-99m, Tellurium-125, Tellurium-132,Thallium-204, Thorium-228, Thorium-232, Thallium-170, Tin-113, Tin-114,Titanium-44, Tungsten-185, Vanadium-48, Vanadium-49, Ytterbium-169,Yttrium-86, Yttrium-88, Yttrium-90, Yttrium-91, Zinc-65, andZirconium-95. Radionuclides useful for imaging include radioisotopes ofcopper (Cu), gallium (Ga), indium (In), rhenium (Rh), and technetium(Tc), including isotopes ⁶⁴Cu, ⁶⁷Cu, ¹¹¹In, ⁹⁹mTc, ⁶⁷Ga or ⁶⁸Ga. Metalsuseful for X-ray contrast agents include radioisotopes of Re, Sm, Ho,Lu, Yt, Pm, Bi, Pd, Gd, La, Au, Yb, Dy, Cu, Rh, Ag and Ir.

A “protein destabilization sequence” or “protein destabilization domain”includes one or more amino acid residues, which, when present at theN-terminus or C-terminus of a protein, reduces or decreases thehalf-life of the linked protein of by at least 80%, preferably at least90%, more preferably at least 95% or more, e.g., 99%, relative to acorresponding protein which lacks the protein destabilization sequenceor domain. A protein destabilization sequence includes, but is notlimited to, a PEST sequence, for example, a PEST sequence from cyclin,e.g., mitotic cyclins, uracil permease or ODC, a sequence from theC-terminal region of a short-lived protein such as ODC, early responseproteins such as cytokines, lymphokines, protooncogenes, e.g., c-myc orc-fos, MyoD, HMG CoA reductase, S-adenosyl methionine decarboxylase, CLsequences, a cyclin destruction box, N-degron, or a protein or afragment thereof which is ubiquitinated in vivo.

I. Mutant Hydrolases and Fusions Thereof

Mutant hydrolases within the scope of the invention include but are notlimited to those prepared via recombinant techniques, e.g.,site-directed mutagenesis or recursive mutagenesis, and comprise one ormore amino acid substitutions which render the mutant hydrolase capableof forming a stable, e.g., covalent, bond with a substrate, such as asubstrate modified to contain one or more functional groups, for acorresponding nonmutant (wild-type) hydrolase which bond is more stablethan the bond formed between a corresponding wild-type hydrolase and thesubstrate. Hydrolases within the scope of the invention include, but arenot limited to, peptidases, esterases (e.g., cholesterol esterase),glycosidases (e.g., glucosamylase), phosphatases (e.g., alkalinephosphatase) and the like. For instance, hydrolases include, but are notlimited to, enzymes acting on ester bonds such as carboxylic esterhydrolases, thiolester hydrolases, phosphoric monoester hydrolases,phosphoric diester hydrolases, triphosphoric monoester hydrolases,sulfuric ester hydrolases, diphosphoric monoester hydrolases, phosphorictriester hydrolases, exodeoxyribonucleases producing5′-phosphomonoesters, exoribonucleases producing 5′-phosphomonoesters,exoribonucleases producing 3′-phosphomonoesters, exonucleases activewith either ribo- or deoxyribonucleic acid, exonucleases active witheither ribo- or deoxyribonucleic acid, endodeoxyribonucleases producing5′-phosphomonoesters, endodeoxyribonucleases producing other than5′-phosphomonoesters, site-specific endodeoxyribonucleases specific foraltered bases, endoribonucleases producing 5′-phosphomonoesters,endoribonucleases producing other than 5′-phosphomonoesters,endoribonucleases active with either ribo- or deoxyribonucleic,endoribonucleases active with either ribo- or deoxyribonucleicglycosylases; glycosidases, e.g., enzymes hydrolyzing O- and S-glycosyl,and hydrolyzing N-glycosyl compounds; acting on ether bonds such astrialkylsulfonium hydrolases or ether hydrolases; enzymes acting onpeptide bonds (peptide hydrolases) such as aminopeptidases,dipeptidases, dipeptidyl-peptidases and tripeptidyl-peptidases,peptidyl-dipeptidases, serine-type carboxypeptidases,metallocarboxypeptidases, cysteine-type carboxypeptidases, omegapeptidases, serine endopeptidases, cysteine endopeptidases, asparticendopeptidases, metalloendopeptidases, threonine endopeptidases, andendopeptidases of unknown catalytic mechanism; enzymes acting oncarbon-nitrogen bonds, other than peptide bonds, such as those in linearamides, in cyclic amides, in linear amidines, in cyclic amidines, innitriles, or other compounds; enzymes acting on acid anhydrides such asthose in phosphorous-containing anhydrides and in sulfonyl-containinganhydrides; enzymes acting on acid anhydrides (catalyzing transmembranemovement); enzymes acting on acid anhydrides or involved in cellular andsubcellular movement; enzymes acting on carbon-carbon bonds (e.g., inketonic substances); enzymes acting on halide bonds (e.g., in C-halidecompounds), enzymes acting on phosphorus-nitrogen bonds; enzymes actingon sulfur-nitrogen bonds; enzymes acting on carbon-phosphorus bonds; andenzymes acting on sulfur-sulfur bonds. Exemplary hydrolases acting onhalide bonds include, but are not limited to, alkylhalidase, 2-haloaciddehalogenase, haloacetate dehalogenase, thyroxine deiodinase, haloalkanedehalogenase, 4-chlorobenzoate dehalogenase, 4-chlorobenzoyl-CoAdehalogenase, and atrazine chlorohydrolase. Exemplary hydrolases thatact on carbon-nitrogen bonds in cyclic amides include, but are notlimited to, barbiturase, dihydropyrimidinase, dihydroorotase,carboxymethylhydantoinase, allantoinase, β-lactamase,imidazolonepropionase, 5-oxoprolinase (ATP-hydrolysing), creatininase,L-lysine-lactamase, 6-aminohexanoate-cyclic-dimer hydrolase,2,5-dioxopiperazine hydrolase, N-methylhydantoinase (ATP-hydrolysing),cyanuric acid amidohydrolase, maleimide hydrolase. “Beta-lactamase” asused herein includes Class A, Class C and Class D beta-lactamases aswell as D-ala carboxypeptidase/transpeptidase, esterase EstB, penicillinbinding protein 2X, penicillin binding protein 5, and D-amino peptidase.Preferably, the beta-lactamase is a serine beta-lactamase, e.g., onehaving a catalytic serine residue at a position corresponding to residue70 in the serine beta-lactamase of S. aureus PC1, and a glutamic acidresidue at a position corresponding to residue 166 in the serinebeta-lactamase of S. aureus PC1, optionally having a lysine residue at aposition corresponding to residue 73, and also optionally having alysine residue at a position corresponding to residue 234, in thebeta-lactamase of S. aureus PC1.

In one embodiment, the mutant hydrolase of the invention comprises atleast one amino acid substitution in a residue which, in the wild-typehydrolase, is associated with activating a water molecule, e.g., aresidue in a catalytic triad or an auxiliary residue, wherein theactivated water molecule cleaves the bond formed between a catalyticresidue in the wild-type hydrolase and a substrate of the hydrolase. Asused herein, an “auxiliary residue” is a residue which alters theactivity of another residue, e.g., it enhances the activity of a residuethat activates a water molecule. Residues which activate water withinthe scope of the invention include but are not limited to those involvedin acid-base catalysis, for instance, histidine, aspartic acid andglutamic acid. In another embodiment, the mutant hydrolase of theinvention comprises at least one amino acid substitution in a residuewhich, in the wild-type hydrolase, forms an ester intermediate bynucleophilic attack of a substrate for the hydrolase. A substrate usefulwith a mutant hydrolase of the invention is one which is specificallybound by a mutant hydrolase, and preferably results in a bond formedwith an amino acid, e.g., the reactive residue, of the mutant hydrolasewhich bond is more stable than the bond formed between the substrate andthe corresponding amino acid of the wild-type hydrolase. While themutant hydrolase specifically binds substrates which may be specificallybound by the corresponding wild-type hydrolase, no product orsubstantially less product, e.g., 2-, 10-, 100-, or 1000-fold less, isformed from the interaction between the mutant hydrolase and thesubstrate under conditions which result in product formation by areaction between the corresponding wild-type hydrolase and substrate.The lack of, or reduced amounts of, product formation by the mutanthydrolase is due to at least one substitution in the mutant hydrolase,which substitution results in the mutant hydrolase forming a bond withthe substrate which is more stable than the bond formed between thecorresponding wild-type hydrolase and the substrate. Preferably, thebond formed between a mutant hydrolase and a substrate of the inventionhas a half-life (i.e., t_(1/2)) that is greater than, e.g., at least2-fold, and more preferably at least 4- or even 10-fold, and up to 100-,1000- or 10.000-fold greater or more, than the t_(1/2) of the bondformed between a corresponding wild-type hydrolase and the substrateunder conditions which result in product formation by the correspondingwild-type hydrolase. Preferably, the bond formed between the mutanthydrolase and the substrate has a t_(1/2) of at least 30 minutes andpreferably at least 4 hours, and up to at least 10 hours, and isresistant to disruption by washing, protein denaturants, and/or hightemperatures, e.g., the bond is stable to boiling in SDS.

In yet another embodiment, the mutant hydrolase of the inventioncomprises at least two amino acid substitutions, one substitution in aresidue which, in the wild-type hydrolase, is associated with activatinga water molecule or in a residue which, in the wild-type hydrolase,forms an ester intermediate by nucleophilic attack of a substrate forthe hydrolase, and another substitution in a residue which, in thewild-type hydrolase, is at or near a binding site(s) for a hydrolasesubstrate, e.g., the residue is within 3 to 5 Å of a hydrolase substratebound to a wild-type hydrolase but is not in a residue that, in thecorresponding wild-type hydrolase, is associated with activating a watermolecule or which forms ester intermediate with a substrate. In oneembodiment, the second substitution is in a residue which, in thewild-type hydrolase lines the site(s) for substrate entry into thecatalytic pocket of the hydrolase, e.g., a residue that is within theactive site cavity and within 3 to 5 Å of a hydrolase substrate bound tothe wild-type hydrolase such as a residue in a tunnel for the substratethat is not a residue in the corresponding wild-type hydrolase which isassociated with activating a water molecule or which forms an esterintermediate with a substrate. The additional substitution(s) preferablyincrease the rate of stable covalent bond formation of those mutants toa substrate of a corresponding wild-type hydrolase. In one embodiment,one substitution is at a residue in the wild-type hydrolase thatactivates the water molecule, e.g., a histidine residue, and is at aposition corresponding to amino acid residue 272 of a Rhodococcusrhodochrous dehalogenase, e.g., the substituted amino acid at theposition corresponding to amino acid residue 272 is phenylalanine orglycine. In another embodiment, one substitution is at a residue in thewild-type hydrolase which forms an ester intermediate with thesubstrate, e.g., an aspartate residue, and at a position correspondingto amino acid residue 106 of a Rhodococcus rhodochrous dehalogenase. Inone embodiment, the second substitution is at an amino acid residuecorresponding to a position 175, 176 or 273 of Rhodococcus rhodochrousdehalogenase, e.g., the substituted amino acid at the positioncorresponding to amino acid residue 175 is methionine, valine,glutamate, aspartate, alanine, leucine, serine or cysteine, thesubstituted amino acid at the position corresponding to amino acidresidue 176 is serine, glycine, asparagine, aspartate, threonine,alanine or arginine, and/or the substituted amino acid at the positioncorresponding to amino acid residue 273 is leucine, methionine orcysteine. In yet another embodiment, the mutant hydrolase furthercomprises a third and optionally a fourth substitution at an amino acidresidue in the wild-type hydrolase that is within the active site cavityand within 3 to 5 Å of a hydrolase substrate bound to the wild-typehydrolase, e.g., the third substitution is at a position correspondingto amino acid residue 175, 176 or 273 of a Rhodococcus rhodochrousdehalogenase, and the fourth substitution is at a position correspondingto amino acid residue 175, 176 or 273 of a Rhodococcus rhodochrousdehalogenase. A mutant hydrolase may include other substitution(s),e.g., those which are introduced to facilitate cloning of thecorresponding gene or a portion thereof, and/or additional residue(s) ator near the N- and/or C-terminus, e.g., those which are introduced tofacilitate cloning of the corresponding gene or a portion thereof butwhich do not necessarily have an activity, e.g., are not separatelydetectable.

For example, wild-type dehalogenase DhaA cleaves carbon-halogen bonds inhalogenated hydrocarbons (HaloC₃-HaloC₁₀). The catalytic center of DhaAis a classic catalytic triad including a nucleophile, an acid and ahistidine residue. The amino acids in the triad are located deep insidethe catalytic pocket of DhaA (about 10 Δ long and about 20 Δ² in crosssection). The halogen atom in a halogenated substrate for DhaA, forinstance, the chlorine atom of a Cl-alkane substrate, is positioned inclose proximity to the catalytic center of DhaA. DhaA binds thesubstrate, likely forms an ES complex, and an ester intermediate isformed by nucleophilic attack of the substrate by Asp106 (the numberingis based on the protein sequence of DhaA) of DhaA. His272 of DhaA thenactivates water and the activated water hydrolyzes the intermediate,releasing product from the catalytic center. As described herein, mutantDhaAs, e.g., a DhaA.H272F mutant, which likely retains the 3-D structurebased on a computer modeling study and basic physico-chemicalcharacteristics of wild-type DhaA (DhaA.WT), were not capable ofhydrolyzing one or more substrates of the wild-type enzyme, e.g., forCl-alkanes, releasing the corresponding alcohol released by thewild-type enzyme. As further described herein, mutant serinebeta-lactamases, e.g., a BlaZ.E166D mutant, a BlaZ.N170Q mutant and aBlaZ.E166D:N170Q mutant, were not capable of hydrolyzing one or moresubstrates of a wild-type serine beta-lactamase.

Thus, in one embodiment of the invention, a mutant hydrolase is a mutantdehalogenase comprising at least one amino acid substitution in aresidue which, in the wild-type dehalogenase, is associated withactivating a water molecule, e.g., a residue in a catalytic triad or anauxiliary residue, wherein the activated water molecule cleaves the bondformed between a catalytic residue in the wild-type dehalogenase and asubstrate of the dehalogenase. In one embodiment, at least onesubstitution is in a residue corresponding to residue 272 in DhaA fromRhodococcus rhodochrous. A “corresponding residue” is a residue whichhas the same activity (function) in one wild-type protein relative to areference wild-type protein and optionally is in the same relativeposition when the primary sequences of the two proteins are aligned. Forexample, a residue which forms part of a catalytic triad and activates awater molecule in one enzyme may be residue 272 in that enzyme, whichresidue 272 corresponds to residue 73 in another enzyme, wherein residue73 forms part of a catalytic triad and activates a water molecule. Thus,in one embodiment, a mutant dehalogenase of the invention has a residueother than histidine, e.g., a phenylalanine residue, at a positioncorresponding to residue 272 in DhaA from Rhodococcus rhodochrous. Inanother embodiment of the invention, a mutant hydrolase is a mutantdehalogenase comprising at least one amino acid substitution in aresidue corresponding to residue 106 in DhaA from Rhodococcusrhodochrous, e.g., a substitution to a residue other than aspartate. Forexample, a mutant dehalogenase of the invention has a cysteine or aglutamate residue at a position corresponding to residue 106 in DhaAfrom Rhodococcus rhodochrous. In a further embodiment, the mutanthydrolase is a mutant dehalogenase comprising at least two amino acidsubstitutions, one in a residue corresponding to residue 106 and one ina residue corresponding to residue 272 in DhaA from Rhodococcusrhodochrous. In one embodiment, the mutant hydrolase is a mutantdehalogenase comprising at least two amino acid substitutions, one in aresidue corresponding to residue 272 in DhaA from Rhodococcusrhodochrous and another in a residue corresponding to residue 175, 176,245 and/or 273 in DhaA from Rhodococcus rhodochrous. In yet a furtherembodiment, the mutant hydrolase is a mutant serine beta-lactamasecomprising at least one amino acid substitution in a residuecorresponding to residue 166 or residue 170 in a serine beta-lactamaseof Staphylococcus aureus PC1.

In one embodiment, the mutant hydrolase is a haloalkane dehalogenase,e.g., such as those found in Gram-negative (Keuning et al., 1985) andGram-positive haloalkane-utilizing bacteria (Keuning et al., 1985;Yokota et al., 1987; Scholtz et al., 1987; Sallis et al., 1990).Haloalkane dehalogenases, including DhlA from Xanthobacter autotrophicusGJ10 (Janssen et al., 1988, 1989), DhaA from Rhodococcus rhodochrous,and LinB from Spingomonas paucimobilis UT26 (Nagata et al., 1997) areenzymes which catalyze hydrolytic dehalogenation of correspondinghydrocarbons. Halogenated aliphatic hydrocarbons subject to conversioninclude C₂-C₁₀ saturated aliphatic hydrocarbons which have one or morehalogen groups attached, wherein at least two of the halogens are onadjacent carbon atoms. Such aliphatic hydrocarbons include volatilechlorinated aliphatic (VCA) hydrocarbons. VCA's include, for example,aliphatic hydrocarbons such as dichloroethane, 1,2-dichloro-propane,1,2-dichlorobutane and 1,2,3-trichloropropane. The term “halogenatedhydrocarbon” as used herein means a halogenated aliphatic hydrocarbon.As used herein the term “halogen” includes chlorine, bromine, iodine,fluorine, astatine and the like. A preferred halogen is chlorine.

In one embodiment, the mutant hydrolase is a thermostable hydrolase suchas a thermostable dehalogenase comprising at least one substitution at aposition corresponding to amino acid residue 117 and/or 175 of aRhodococcus rhodochrous dehalogenase, which substitution is correlatedwith enhanced thermostability. In one embodiment, the thermostablehydrolase is capable of binding a hydrolase substrate at lowtemperatures, e.g., from 0° C. to about 25° C. In one embodiment, athermostable hydrolase is a thermostable mutant hydrolase, i.e., onehaving one or more substitutions in addition to the substitution at aposition corresponding to amino acid residue 117 and/or 175 of aRhodococcus rhodochrous dehalogenase. In one embodiment, a thermostablemutant dehalogenase has a substitution which results in removal of acharged residue, e.g., lysine. In one embodiment, a thermostable mutantdehalogenase has a serine or methionine at a position corresponding toresidue 117 and/or 175 in DhaA from Rhodococcus rhodochrous.

The invention also provides a fusion protein comprising a mutanthydrolase and amino acid sequences for a protein or peptide of interest,e.g., sequences for a marker protein, e.g., a selectable marker protein,affinity tag, e.g., a polyhistidine sequence, an enzyme of interest,e.g., luciferase, RNasin, RNase, and/or GFP, a nucleic acid bindingprotein, an extracellular matrix protein, a secreted protein, anantibody or a portion thereof such as Fc, a bioluminescence protein, areceptor ligand, a regulatory protein, a serum protein, an immunogenicprotein, a fluorescent protein, a protein with reactive cysteines, areceptor protein, e.g., NMDA receptor, a channel protein, e.g., an ionchannel protein such as a sodium-, potassium- or a calcium-sensitivechannel protein including a HERG channel protein, a membrane protein, acytosolic protein, a nuclear protein, a structural protein, aphosphoprotein, a kinase, a signaling protein, a metabolic protein, amitochondrial protein, a receptor associated protein, a fluorescentprotein, an enzyme substrate, e.g., a protease substrate, atranscription factor, a protein destabilization sequence, or atransporter protein, e.g., EAAT1-4 glutamate transporter, as well astargeting signals, e.g., a plastid targeting signal, such as amitochondrial localization sequence, a nuclear localization signal or amyristilation sequence, that directs the mutant hydrolase to aparticular location.

The fusion protein may be expressed from a recombinant DNA which encodesthe mutant hydrolase and at least one protein of interest, or formed bychemical synthesis. The protein of interest may be fused to theN-terminus or the C-terminus of the mutant hydrolase. In one embodiment,the fusion protein comprises a protein of interest at the N-terminus,and another protein, e.g., a different protein, at the C-terminus, ofthe mutant hydrolase. For example, the protein of interest may be afluorescent protein or an antibody. Optionally, the proteins in thefusion are separated by a connector sequence, e.g., preferably onehaving at least 2 amino acid residues, such as one having 13 to 17 aminoacid residues. The presence of a connector sequence in a fusion proteinof the invention does not substantially alter the function of eitherprotein in the fusion relative to the function of each individualprotein. Thus, for a fusion of a mutant dehalogenase and Renillaluciferase, the presence of a connector sequence does not substantiallyalter the stability of the bond formed between the mutant dehalogenaseand a substrate therefor or the activity of the luciferase. For anyparticular combination of proteins in a fusion, a wide variety ofconnector sequences may be employed. In one embodiment, the connectorsequence is a sequence recognized by an enzyme, e.g., a cleavablesequence. For instance, the connector sequence may be one recognized bya caspase, e.g., DEVD (SEQ ID NO:17), or is a photocleavable sequence.

In one embodiment, the fusion protein may comprise a protein of interestat the N-terminus and, optionally, a different protein of interest atthe C-terminus of the mutant hydrolase. As described herein, fusions ofa mutant DhaA with GST (at the N-terminus), a Flag sequence (at theC-terminus) and Renilla luciferase (at the N-terminus or C-terminus) hadno detectable effect on bond formation between the mutant DhaA and asubstrate for wild-type DhaA which includes a functional group.Moreover, a fusion of a Flag sequence and DhaA.H272F could be attachedto a solid support via a streptavidin-biotin-C₁₀H₂₁N₁O₂—Cl-DhaA.H272Fbridge (an SFlag-ELISA experiment).

In one embodiment, a fusion protein includes a mutant hydrolase and aprotein that is associated with a membrane or a portion thereof, e.g.,targeting proteins such as those for endoplasmic reticulum targeting,cell membrane bound proteins, e.g., an integrin protein or a domainthereof such as the cytoplasmic, transmembrane and/or extracellularstalk domain of an integrin protein, and/or a protein that links themutant hydrolase to the cell surface, e.g., a glycosylphosphoinositolsignal sequence.

II. Optimized Hydrolase Sequences, and Vectors and Host Cells Encodingthe Hydrolase

Also provided is an isolated nucleic acid molecule (polynucleotide)comprising a nucleic acid sequence encoding a hydrolase or a fusionthereof. In one embodiment, the isolated nucleic acid molecule comprisesa nucleic acid sequence which is optimized for expression in at leastone selected host. Optimized sequences include sequences which are codonoptimized, i.e., codons which are employed more frequently in oneorganism relative to another organism, e.g., a distantly relatedorganism, as well as modifications to add or modify Kozak sequencesand/or introns, and/or to remove undesirable sequences, for instance,potential transcription factor binding sites. In one embodiment, thepolynucleotide includes a nucleic acid sequence encoding a dehalogenase,which nucleic acid sequence is optimized for expression is a selectedhost cell. In one embodiment, the optimized polynucleotide no longerhybridizes to the corresponding non-optimized sequence, e.g., does nothybridize to the non-optimized sequence under medium or high stringencyconditions. In another embodiment, the polynucleotide has less than 90%,e.g., less than 80%, nucleic acid sequence identity to the correspondingnon-optimized sequence and optionally encodes a polypeptide having atleast 80%, e.g., at least 85%, 90% or more, amino acid sequence identitywith the polypeptide encoded by the non-optimized sequence. Constructs,e.g., expression cassettes, and vectors comprising the isolated nucleicacid molecule, as well as kits comprising the isolated nucleic acidmolecule, construct or vector are also provided.

A nucleic acid molecule comprising a nucleic acid sequence encoding afusion with hydrolase is optionally optimized for expression in aparticular host cell and also optionally operably linked totranscription regulatory sequences, e.g., one or more enhancers, apromoter, a transcription termination sequence or a combination thereof,to form an expression cassette.

In one embodiment, a nucleic acid sequence encoding a hydrolase or afusion thereof is optimized by replacing codons in a wild-type or mutanthydrolase sequence with codons which are preferentially employed in aparticular (selected) cell. Preferred codons have a relatively highcodon usage frequency in a selected cell, and preferably theirintroduction results in the introduction of relatively few transcriptionfactor binding sites for transcription factors present in the selectedhost cell, and relatively few other undesirable structural attributes.Thus, the optimized nucleic acid product has an improved level ofexpression due to improved codon usage frequency, and a reduced risk ofinappropriate transcriptional behavior due to a reduced number ofundesirable transcription regulatory sequences.

An isolated and optimized nucleic acid molecule of the invention mayhave a codon composition that differs from that of the correspondingwild-type nucleic acid sequence at more than 30%, 35%, 40% or more than45%, e.g., 50%, 55%, 60% or more of the codons. Preferred codons for usein the invention are those which are employed more frequently than atleast one other codon for the same amino acid in a particular organismand, more preferably, are also not low-usage codons in that organism andare not low-usage codons in the organism used to clone or screen for theexpression of the nucleic acid molecule. Moreover, preferred codons forcertain amino acids (i.e., those amino acids that have three or morecodons), may include two or more codons that are employed morefrequently than the other (non-preferred) codon(s). The presence ofcodons in the nucleic acid molecule that are employed more frequently inone organism than in another organism results in a nucleic acid moleculewhich, when introduced into the cells of the organism that employs thosecodons more frequently, is expressed in those cells at a level that isgreater than the expression of the wild-type or parent nucleic acidsequence in those cells.

In one embodiment of the invention, the codons that are different arethose employed more frequently in a mammal, while in another embodimentthe codons that are different are those employed more frequently in aplant. Preferred codons for different organisms are known to the art,e.g., see www.kazusa.or.jp./codon/. A particular type of mammal, e.g., ahuman, may have a different set of preferred codons than another type ofmammal. Likewise, a particular type of plant may have a different set ofpreferred codons than another type of plant. In one embodiment of theinvention, the majority of the codons that differ are ones that arepreferred codons in a desired host cell. Preferred codons for organismsincluding mammals (e.g., humans) and plants are known to the art (e.g.,Wada et al., 1990; Ausubel et al., 1997). For example, preferred humancodons include, but are not limited to, CGC (Arg), CTG (Leu), TCT (Ser),AGC (Ser), ACC (Thr), CCA (Pro), CCT (Pro), GCC (Ala), GGC (Gly), GTG(Val), ATC (Ile), ATT (Ile), AAG (Lys), AAC (Asn), CAG (Gln), CAC (His),GAG (Glu), GAC (Asp), TAC (Tyr), TGC (Cys) and TTC (Phe) (Wada et al.,1990). Thus, in one embodiment, synthetic nucleic acid molecules of theinvention have a codon composition which differs from a wild typenucleic acid sequence by having an increased number of the preferredhuman codons, e.g., CGC, CTG, TCT, AGC, ACC, CCA, CCT, GCC, GGC, GTG,ATC, ATT, AAG, AAC, CAG, CAC, GAG, GAC, TAC, TGC, TTC, or anycombination thereof. For example, the nucleic acid molecule of theinvention may have an increased number of CTG or TTG leucine-encodingcodons, GTG or GTC valine-encoding codons, GGC or GGT glycine-encodingcodons, ATC or ATT isoleucine-encoding codons, CCA or CCTproline-encoding codons, CGC or CGT arginine-encoding codons, AGC or TCTserine-encoding codons, ACC or ACT threonine-encoding codon, GCC or GCTalanine-encoding codons, or any combination thereof, relative to thewild-type nucleic acid sequence. In another embodiment, preferred C.elegans codons include, but are not limited, to UUC (Phe), UUU (Phe),CUU (Leu), UUG (Leu), AUU (Ile), GUU (Val), GUG (Val), UCA (Ser), UCU(Ser), CCA (Pro), ACA (Thr), ACU (Thr), GCU (Ala), GCA (Ala), UAU (Tyr),CAU (His), CAA (Gln), AAU (Asn), AAA (Lys), GAU (Asp), GAA (Glu), UGU(Cys), AGA (Arg), CGA (Arg), CGU (Arg), GGA (Gly), or any combinationthereof. In yet another embodiment, preferred Drosophilia codonsinclude, but are not limited to, UUC (Phe), CUG (Leu), CUC (Leu), AUC(Ile), AUU (Ile), GUG (Val), GUC (Val), AGC (Ser), UCC (Ser), CCC (Pro),CCG (Pro), ACC (Thr), ACG (Thr), GCC (Ala), GCU (Ala), UAC (Tyr), CAC(His), CAG (Gln), AAC (Asn), AAG (Lys), GAU (Asp), GAG (Glu), UGC (Cys),CGC (Arg), GGC (Gly), GGA (gly), or any combination thereof. Preferredyeast codons include but are not limited to UUU (Phe), UUG (Leu), UUA(Leu), CCU (Leu), AUU (Ile), GUU (Val), UCU (Ser), UCA (Ser), CCA (Pro),CCU (Pro), ACU (Thr), ACA (Thr), GCU (Ala), GCA (Ala), UAU (Tyr), UAC(Tyr), CAU (His), CAA (Gln), AAU (Asn), AAC (Asn), AAA (Lys), AAG (Lys),GAU (Asp), GAA (Glu), GAG (Glu), UGU (Cys), CGU (Trp), AGA (Arg), CGU(Arg), GGU (Gly), GGA (Gly), or any combination thereof. Similarly,nucleic acid molecules having an increased number of codons that areemployed more frequently in plants, have a codon composition whichdiffers from a wild-type or parent nucleic acid sequence by having anincreased number of the plant codons including, but not limited to, CGC(Arg), CTT (Leu), TCT (Ser), TCC (Ser), ACC (Thr), CCA (Pro), CCT (Pro),GCT (Ser), GGA (Gly), GTG (Val), ATC (Ile), ATT (Ile), AAG (Lys), AAC(Asn), CAA (Gln), CAC (His), GAG (Glu), GAC (Asp), TAC (Tyr), TGC (Cys),TTC (Phe), or any combination thereof (Murray et al., 1989). Preferredcodons may differ for different types of plants (Wada et al., 1990).

In one embodiment, an optimized nucleic acid sequence encoding ahydrolase or fusion thereof has less than 100%, e.g., less than 90% orless than 80%, nucleic acid sequence identity relative to anon-optimized nucleic acid sequence encoding a corresponding hydrolaseor fusion thereof. For instance, an optimized nucleic acid sequenceencoding DhaA has less than about 80% nucleic acid sequence identityrelative to non-optimized (wild-type) nucleic acid sequence encoding acorresponding DhaA, and the DhaA encoded by the optimized nucleic acidsequence optionally has at least 85% amino acid sequence identity to acorresponding wild-type DhaA. In one embodiment, the activity of a DhaAencoded by the optimized nucleic acid sequence is at least 10%, e.g.,50% or more, of the activity of a DhaA encoded by the non-optimizedsequence, e.g., a mutant DhaA encoded by the optimized nucleic acidsequence binds a substrate with substantially the same efficiency, i.e.,at least 50%, 80%, 100% or more, as the mutant DhaA encoded by thenon-optimized nucleic acid sequence binds the same substrate.

An exemplary optimized DhaA gene has the following sequence:

hDhaA.v2.1-6F (FINAL, with flanking sequences)

(SEQ ID NO: 50) NNNNGCTAGCCAGCTGGCgcgGATATCGCCACCATGGGATCCGAGATTGGGACAGGGTTcCCTTTTGATCCTCAcTATGTtGAaGTGCTGGGgGAaAGAATGCAcTAcGTGGATGTGGGGCCTAGAGATGGGACcCCaGTGCTGTTcCTcCAcGGGAAcCCTACATCTagcTAcCTGTGGAGaAAtATTATaCCTCATGTtGCTCCTagtCATAGgTGcATTGCTCCTGATCTGATcGGGATGGGGAAGTCTGATAAGCCTGActtaGAcTAcTTTTTTGATGAtCATGTtcGATActTGGATGCTTTcATTGAGGCTCTGGGGCTGGAGGAGGTGGTGCTGGTGATaCAcGAcTGGGGGTCTGCTCTGGGGTTTCAcTGGGCTAAaAGgAATCCgGAGAGAGTGAAGGGGATTGCTTGcATGGAgTTTATTcGACCTATTCCTACtTGGGAtGAaTGGCCaGAGTTTGCcAGAGAGACATTTCAaGCcTTTAGAACtGCcGATGTGGGcAGgGAGCTGATTATaGAcCAGAATGCTTTcATcGAGGGGGCTCTGCCTAAaTGTGTaGTcAGACCTCTcACtGAaGTaGAGATGGAcCATTATAGAGAGCCcTTTCTGAAGCCTGTGGATcGcGAGCCTCTGTGGAGgTTtCCaAATGAGCTGCCTATTGCTGGGGAGCCTGCTAATATTGTGGCTCTGGTGGAaGCcTATATGAAcTGGCTGCATCAGagTCCaGTGCCcAAGCTaCTcTTTTGGGGGACtCCgGGaGTtCTGATTCCTCCTGCcGAGGCTGCTAGACTGGCTGAaTCcCTGCCcAAtTGTAAGACcGTGGAcATcGGcCCtGGgCTGTTTTAcCTcCAaGAGGAcAAcCCTGATCTcATcGGGTCTGAGATcGCacGgTGGCTGCCCGGGCTGGCCGGCTAATAGTTAATTAAGTAgG CGGCCGCNNNN.

The nucleic acid molecule or expression cassette may be introduced to avector, e.g., a plasmid or viral vector, which optionally includes aselectable marker gene, and the vector introduced to a cell of interest,for example, a prokaryotic cell such as E. coli, Streptomyces spp.,Bacillus spp., Staphylococcus spp. and the like, as well as eukaryoticcells including a plant (dicot or monocot), fungus, yeast, e.g., Pichia,Saccharomyces or Schizosaccharomyces, or mammalian cell. Preferredmammalian cells include bovine, caprine, ovine, canine, feline,non-human primate, e.g., simian, and human cells. Preferred mammaliancell lines include, but are not limited to, CHO, COS, 293, Hela, CV-1,SH-SY5Y (human neuroblastoma cells), HEK293, and NIH3T3 cells.

The expression of the encoded mutant hydrolase may be controlled by anypromoter capable of expression in prokaryotic cells or eukaryotic cells.Preferred prokaryotic promoters include, but are not limited to, SP6,T7, T5, tac, bla, trp, gal, lac or maltose promoters. Preferredeukaryotic promoters include, but are not limited to, constitutivepromoters, e.g., viral promoters such as CMV, SV40 and RSV promoters, aswell as regulatable promoters, e.g., an inducible or repressiblepromoter such as the tet promoter, the hsp70 promoter and a syntheticpromoter regulated by CRE. Preferred vectors for bacterial expressioninclude pGEX-5X-3, and for eukaryotic expression include pCIneo-CMV.

The nucleic acid molecule, expression cassette and/or vector of theinvention may be introduced to a cell by any method including, but notlimited to, calcium-mediated transformation, electroporation,microinjection, lipofection, particle bombardment and the like.

III. Functional Groups

Functional groups useful in the substrates and methods of the inventionare molecules that are detectable or capable of detection. A functionalgroup within the scope of the invention is capable of being covalentlylinked to one reactive substituent of a bifunctional linker or asubstrate for a hydrolase, and, as part of a substrate of the invention,has substantially the same activity as a functional group which is notlinked to a substrate found in nature and is capable of forming a stablecomplex with a mutant hydrolase. Functional groups thus have one or moreproperties that facilitate detection, and optionally the isolation, ofstable complexes between a substrate having that functional group and amutant hydrolase. For instance, functional groups include those with acharacteristic electromagnetic spectral property such as emission orabsorbance, magnetism, electron spin resonance, electrical capacitance,dielectric constant or electrical conductivity as well as functionalgroups which are ferromagnetic, paramagnetic, diamagnetic, luminescent,electrochemiluminescent, fluorescent, phosphorescent, chromatic,antigenic, or have a distinctive mass. A functional group includes, butis not limited to, a nucleic acid molecule, i.e., DNA or RNA, e.g., anoligonucleotide or nucleotide, such as one having nucleotide analogs,DNA which is capable of binding a protein, single stranded DNAcorresponding to a gene of interest, RNA corresponding to a gene ofinterest, mRNA which lacks a stop codon, an aminoacylated initiatortRNA, an aminoacylated amber suppressor tRNA, or double stranded RNA forRNAi, a protein, e.g., a luminescent protein, a peptide, a peptidenucleic acid, an epitope recognized by a ligand, e.g., biotin orstreptavidin, a hapten, an amino acid, a lipid, a lipid bilayer, a solidsupport, a fluorophore, a chromophore, a reporter molecule, aradionuclide, such as a radioisotope for use in, for instance,radioactive measurements or a stable isotope for use in methods such asisotope coded affinity tag (ICAT), an electron opaque molecule, an X-raycontrast reagent, a MRI contrast agent, e.g., manganese, gadolinium(III) or iron-oxide particles, and the like. In one embodiment, thefunctional group is an amino acid, protein, glycoprotein,polysaccharide, triplet sensitizer, e.g., CALI, nucleic acid molecule,drug, toxin, lipid, biotin, or solid support, such as self-assembledmonolayers (see, e.g., Kwon et al., 2004), binds Ca²⁺, binds K⁺, bindsNa⁺, is pH sensitive, is electron opaque, is a chromophore, is a MRIcontrast agent, fluoresces in the presence of NO or is sensitive to areactive oxygen, a nanoparticle, an enzyme, a substrate for an enzyme,an inhibitor of an enzyme, for instance, a suicide substrate (see, e.g.,Kwon et al., 2004), a cofactor, e.g., NADP, a coenzyme, a succinimidylester or aldehyde, luciferin, glutathione, NTA, biotin, cAMP,phosphatidylinositol, a ligand for cAMP, a metal, a nitroxide or nitronefor use as a spin trap (detected by electron spin resonance (ESR), ametal chelator, e.g., for use as a contrast agent, in time resolvedfluorescence or to capture metals, a photocaged compound, e.g., whereirradiation liberates the caged compound such as a fluorophore, anintercalator, e.g., such as psoralen or another intercalator useful tobind DNA or as a photoactivatable molecule, a triphosphate or aphosphoramidite, e.g., to allow for incorporation of the substrate intoDNA or RNA, an antibody, or a heterobifunctional cross-linker such asone useful to conjugate proteins or other molecules, cross-linkersincluding but not limited to hydrazide, aryl azide, maleimide,iodoacetamide/bromoacetamide, N-hydroxysuccinimidyl ester, mixeddisulfide such as pyridyl disulfide, glyoxal/phenylglyoxal, vinylsulfone/vinyl sulfonamide, acrylamide, boronic ester, hydroxamic acid,imidate ester, isocyanate/isothiocyanate, orchlorotriazine/dichlorotriazine.

For instance, a functional group includes but is not limited to one ormore amino acids, e.g., a naturally occurring amino acid or anon-natural amino acid, a peptide or polypeptide (protein) including anantibody or a fragment thereof, a His-tag, a FLAG tag, a Strep-tag, anenzyme, a cofactor, a coenzyme, a peptide or protein substrate for anenzyme, for instance, a branched peptide substrate (e.g., Z-aminobenzoyl(Abz)-Gly-Pro-Ala-Leu-Ala-4-nitrobenzyl amide (NBA), a suicidesubstrate, or a receptor, one or more nucleotides (e.g., ATP, ADP, AMP,GTP or GDP) including analogs thereof, e.g., an oligonucleotide, doublestranded or single stranded DNA corresponding to a gene or a portionthereof, e.g., DNA capable of binding a protein such as a transcriptionfactor, RNA corresponding to a gene, for instance, mRNA which lacks astop codon, or a portion thereof, double stranded RNA for RNAi orvectors therefor, a glycoprotein, a polysaccharide, a peptide-nucleicacid (PNA), lipids including lipid bilayers; or is a solid support,e.g., a sedimental particle such as a magnetic particle, a sepharose orcellulose bead, a membrane, glass, e.g., glass slides, cellulose,alginate, plastic or other synthetically prepared polymer, e.g., aneppendorf tube or a well of a multi-well plate, self assembledmonolayers, a surface plasmon resonance chip, or a solid support with anelectron conducting surface, and includes a drug, for instance, achemotherapeutic such as doxorubicin, 5-fluorouracil, or camptosar(CPT-11; Irinotecan), an aminoacylated tRNA such as an aminoacylatedinitiator tRNA or an aminoacylated amber suppressor tRNA, a moleculewhich binds Ca²⁺, a molecule which binds K⁺, a molecule which binds Na⁺,a molecule which is pH sensitive, a radionuclide, a molecule which iselectron opaque, a contrast agent, e.g., barium, iodine or other MRI orX-ray contrast agent, a molecule which fluoresces in the presence of NOor is sensitive to a reactive oxygen, a nanoparticle, e.g., animmunogold particle, paramagnetic nanoparticle, upconvertingnanoparticle, or a quantum dot, a nonprotein substrate for an enzyme, aninhibitor of an enzyme, either a reversible or irreversible inhibitor, achelating agent, a cross-linking group, for example, a succinimidylester or aldehyde, glutathione, biotin or other avidin binding molecule,avidin, streptavidin, cAMP, phosphatidylinositol, heme, a ligand forcAMP, a metal, NTA, and, in one embodiment, includes one or more dyes,e.g., a xanthene dye, a calcium sensitive dye, e.g.,1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)-phenoxy]-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraaceticacid (Fluo-3), a sodium sensitive dye, e.g., 1,3-benzenedicarboxylicacid,4,4′-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis(PBFI),a NO sensitive dye, e.g., 4-amino-5-methylamino-2′,7′-difluorescein, orother fluorophore. In one embodiment, the functional group is a haptenor an immunogenic molecule, i.e., one which is bound by antibodiesspecific for that molecule. In one embodiment, the functional group isnot a radionuclide. In another embodiment, the functional group is aradionuclide, e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I, including a moleculeuseful in diagnostic methods.

Methods to detect a particular functional group are known to the art.For example, a nucleic acid molecule can be detected by hybridization,amplification, binding to a nucleic acid binding protein specific forthe nucleic acid molecule, enzymatic assays (e.g., if the nucleic acidmolecule is a ribozyme), or, if the nucleic acid molecule itselfcomprises a molecule which is detectable or capable of detection, forinstance, a radiolabel or biotin, it can be detected by an assaysuitable for that molecule.

Exemplary functional groups include haptens, e.g., molecules useful toenhance immunogenicity such as keyhole limpet hemacyanin (KLH),cleavable labels, for instance, photocleavable biotin, and fluorescentlabels, e.g., N-hydroxysuccinimide (NHS) modified coumarin andsuccinimide or sulfonosuccinimide modified BODIPY (which can be detectedby UV and/or visible excited fluorescence detection), rhodamine, e.g.,R110, rhodols, CRG6, Texas Methyl Red (carboxytetramethylrhodamine),5-carboxy-X-rhodamine, or fluoroscein, coumarin derivatives, e.g., 7aminocoumarin, and 7-hydroxycoumarin, 2-amino-4-methoxynapthalene,1-hydroxypyrene, resorufin, phenalenones or benzphenalenones (U.S. Pat.No. 4,812,409), acridinones (U.S. Pat. No. 4,810,636), anthracenes, andderivatives of α- and β-napthol, fluorinated xanthene derivativesincluding fluorinated fluoresceins and rhodols (e.g., U.S. Pat. No.6,162,931), bioluminescent molecules, e.g., luciferin, coelenterazine,luciferase, chemiluminescent molecules, e.g., stabilized dioxetanes, andelectrochemiluminescent molecules. A fluorescent (or luminescent)functional group linked to a mutant hydrolase by virtue of being linkedto a substrate for a corresponding wild-type hydrolase, may be used tosense changes in a system, like phosphorylation, in real time. Moreover,a fluorescent molecule, such as a chemosensor of metal ions, e.g., a9-carbonylanthracene modified glycyl-histidyl-lysine (GHK) for Cu²⁺, ina substrate of the invention may be employed to label proteins whichbind the substrate. A luminescent or fluorescent functional group suchas BODIPY, rhodamine green, GFP, or infrared dyes, also finds use as afunctional group and may, for instance, be employed in interactionstudies, e.g., using BRET, FRET, LRET or electrophoresis.

Another class of functional group is a molecule that selectivelyinteracts with molecules containing acceptor groups (an “affinity”molecule). Thus, a substrate for a hydrolase which includes an affinitymolecule can facilitate the separation of complexes having such asubstrate and a mutant hydrolase, because of the selective interactionof the affinity molecule with another molecule, e.g., an acceptormolecule, that may be biological or non-biological in origin. Forexample, the specific molecule with which the affinity moleculeinteracts (referred to as the acceptor molecule) could be a smallorganic molecule, a chemical group such as a sulfhydryl group (—SH) or alarge biomolecule such as an antibody or other naturally occurringligand for the affinity molecule. The binding is normally chemical innature and may involve the formation of covalent or non-covalent bondsor interactions such as ionic or hydrogen bonding. The acceptor moleculemight be free in solution or itself bound to a solid or semi-solidsurface, a polymer matrix, or reside on the surface of a solid orsemi-solid substrate. The interaction may also be triggered by anexternal agent such as light, temperature, pressure or the addition of achemical or biological molecule that acts as a catalyst. The detectionand/or separation of the complex from the reaction mixture occursbecause of the interaction, normally a type of binding, between theaffinity molecule and the acceptor molecule.

Examples of affinity molecules include molecules such as immunogenicmolecules, e.g., epitopes of proteins, peptides, carbohydrates orlipids, i.e., any molecule which is useful to prepare antibodiesspecific for that molecule; biotin, avidin, streptavidin, andderivatives thereof; metal binding molecules; and fragments andcombinations of these molecules. Exemplary affinity molecules includeHis5 (HHHHH) (SEQ ID NO:19), His X6 (HHHHHH) (SEQ ID NO:20), C-myc(EQKLISEEDL) (SEQ ID NO:21), Flag (DYKDDDDK) (SEQ ID NO:22), SteptTag(WSHPQFEK) (SEQ ID NO:23), HA Tag (YPYDVPDYA) (SEQ ID NO:24),thioredoxin, cellulose binding domain, chitin binding domain, S-peptide,T7 peptide, calmodulin binding peptide, C-end RNA tag, metal bindingdomains, metal binding reactive groups, amino acid reactive groups,inteins, biotin, streptavidin, and maltose binding protein. For example,a substrate for a hydrolase which includes biotin is contacted with amutant hydrolase. The presence of the biotin in a complex between themutant hydrolase and the substrate permits selective binding of thecomplex to avidin molecules, e.g., streptavidin molecules coated onto asurface, e.g., beads, microwells, nitrocellulose and the like. Suitablesurfaces include resins for chromatographic separation, plastics such astissue culture surfaces or binding plates, microtiter dishes and beads,ceramics and glasses, particles including magnetic particles, polymersand other matrices. The treated surface is washed with, for example,phosphate buffered saline (PBS), to remove molecules that lack biotinand the biotin-containing complexes isolated. In some case thesematerials may be part of biomolecular sensing devices such as opticalfibers, chemfets, and plasmon detectors.

Another example of an affinity molecule is dansyllysine. Antibodieswhich interact with the dansyl ring are commercially available (SigmaChemical; St. Louis, Mo.) or can be prepared using known protocols suchas described in Antibodies: A Laboratory Manual (Harlow and Lane, 1988).For example, the anti-dansyl antibody is immobilized onto the packingmaterial of a chromatographic column. This method, affinity columnchromatography, accomplishes separation by causing the complex between amutant hydrolase and a substrate of the invention to be retained on thecolumn due to its interaction with the immobilized antibody, while othermolecules pass through the column. The complex may then be released bydisrupting the antibody-antigen interaction. Specific chromatographiccolumn materials such as ion-exchange or affinity Sepharose, Sephacryl,Sephadex and other chromatography resins are commercially available(Sigma Chemical; St. Louis, Mo.; Pharmacia Biotech; Piscataway, N.J.).Dansyllysine may conveniently be detected because of its fluorescentproperties.

When employing an antibody as an acceptor molecule, separation can alsobe performed through other biochemical separation methods such asimmunoprecipitation and immobilization of antibodies on filters or othersurfaces such as beads, plates or resins. For example, complexes of amutant hydrolase and a substrate of the invention may be isolated bycoating magnetic beads with an affinity molecule-specific or ahydrolase-specific antibody. Beads are oftentimes separated from themixture using magnetic fields.

Another class of functional molecules includes molecules detectableusing electromagnetic radiation and includes but is not limited toxanthene fluorophores, dansyl fluorophores, coumarins and coumarinderivatives, fluorescent acridinium moieties, benzopyrene basedfluorophores, as well as 7-nitrobenz-2-oxa-1,3-diazole, and3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diamino-propionic acid.Preferably, the fluorescent molecule has a high quantum yield offluorescence at a wavelength different from native amino acids and morepreferably has high quantum yield of fluorescence that can be excited inthe visible, or in both the UV and visible, portion of the spectrum.Upon excitation at a preselected wavelength, the molecule is detectableat low concentrations either visually or using conventional fluorescencedetection methods. Electrochemiluminescent molecules such as rutheniumchelates and its derivatives or nitroxide amino acids and theirderivatives are detectable at femtomolar ranges and below.

In one embodiment, an optionally detectable functional group includesone of:

wherein R₁ is C₁-C₈.

In addition to fluorescent molecules, a variety of molecules withphysical properties based on the interaction and response of themolecule to electromagnetic fields and radiation can be used to detectcomplexes between a mutant hydrolase and a substrate of the invention.These properties include absorption in the UV, visible and infraredregions of the electromagnetic spectrum, presence of chromophores whichare Raman active, and can be further enhanced by resonance Ramanspectroscopy, electron spin resonance activity and nuclear magneticresonances and molecular mass, e.g., via a mass spectrometer.

Methods to detect and/or isolate complexes having affinity moleculesinclude chromatographic techniques including gel filtration,fast-pressure or high-pressure liquid chromatography, reverse-phasechromatography, affinity chromatography and ion exchange chromatography.Other methods of protein separation are also useful for detection andsubsequent isolation of complexes between a mutant hydrolase and asubstrate of the invention, for example, electrophoresis, isoelectricfocusing and mass spectrometry.

IV. Linkers

The term “linker”, which is also identified by the symbol >L=, refers toa group or groups that covalently attach one or more functional groupsto a substrate which includes a reactive group or to a reactive group. Alinker, as used herein, is not a single covalent bond. The structure ofthe linker is not crucial, provided it yields a substrate that can bebound by its target enzyme. In one embodiment, the linker can be adivalent group that separates a functional group (R) and the reactivegroup by about 5 angstroms to about 1000 angstroms, inclusive, inlength. Other suitable linkers include linkers that separate R and thereactive group by about 5 angstroms to about 100 angstroms, as well aslinkers that separate R and the substrate by about 5 angstroms to about50 angstroms, by about 5 angstroms to about 25 angstroms, by about 5angstroms to about 500 angstroms, or by about 30 angstroms to about 100angstroms.

In one embodiment the linker is an amino acid.

In another embodiment, the linker is a peptide.

In another embodiment, the linker is a divalent branched or unbranchedcarbon chain comprising from about 2 to about 30 carbon atoms, whichchain optionally includes one or more (e.g., 1, 2, 3, or 4) double ortriple bonds, and which chain is optionally substituted with one or more(e.g., 2, 3, or 4) hydroxy or oxo (═O) groups, wherein one or more(e.g., 1, 2, 3, or 4) of the carbon atoms in the chain is optionallyreplaced with a non-peroxide —O—, —S— or —NH— and wherein one or more(e.g., 1, 2, 3, or 4) of the carbon atoms in the chain is replaced withan aryl or heteroaryl ring.

In another embodiment, the linker is a divalent branched or unbranchedcarbon chain comprising from about 2 to about 30 carbon atoms, whichchain optionally includes one or more (e.g., 1, 2, 3, or 4) double ortriple bonds, and which chain is optionally substituted with one or more(e.g., 2, 3, or 4) hydroxy or oxo (═O) groups, wherein one or more(e.g., 1, 2, 3, or 4) of the carbon atoms in the chain is replaced witha non-peroxide —O—, —S— or —NH— and wherein one or more (e.g., 1, 2, 3,or 4) of the carbon atoms in the chain is replaced with one or more(e.g., 1, 2, 3, or 4) aryl or heteroaryl rings.

In another embodiment, the linker is a divalent branched or unbranchedcarbon chain comprising from about 2 to about 30 carbon atoms, whichchain optionally includes one or more (e.g., 1, 2, 3, or 4) double ortriple bonds, and which chain is optionally substituted with one or more(e.g., 2, 3, or 4) hydroxy or oxo (═O) groups, wherein one or more(e.g., 1, 2, 3, or 4) of the carbon atoms in the chain is replaced witha non-peroxide —O—, —S— or —NH— and wherein one or more (e.g., 1, 2, 3,or 4) of the carbon atoms in the chain is replaced with one or more(e.g., 1, 2, 3, or 4) heteroaryl rings.

In another embodiment, the linker is a divalent branched or unbranchedcarbon chain comprising from about 2 to about 30 carbon atoms, whichchain optionally includes one or more (e.g., 1, 2, 3, or 4) double ortriple bonds, and which chain is optionally substituted with one or more(e.g., 2, 3, or 4) hydroxy or oxo (═O) groups, wherein one or more(e.g., 1, 2, 3, or 4) of the carbon atoms in the chain is optionallyreplaced with a non-peroxide —O—, —S— or —NH—.

In another embodiment, the linker is a divalent group of the formula—W—F—W— wherein F is (C₁-C₃₀)alkyl, (C₂-C₃₀)alkenyl, (C₂-C₃₀)alkynyl,(C₃-C₈)cycloalkyl, or (C₆-C₁₀), wherein W is —N(O)C(═O)—, —C(═O)N(O)—,—OC(═O)—, —C(═O)O—, —O—, —S—, —S(O)—, —S(O)₂—, —N(O)—, —C(═O)—, or adirect bond; wherein each Q is independently H or (C₁-C₆)alkyl

In another embodiment, the linker is a divalent branched or unbranchedcarbon chain comprising from about 2 to about 30 carbon atoms, whichchain optionally includes one or more (e.g., 1, 2, 3, or 4) double ortriple bonds, and which chain is optionally substituted with one or more(e.g., 2, 3, or 4) hydroxy or oxo (═O) groups.

In another embodiment, the linker is a divalent branched or unbranchedcarbon chain comprising from about 2 to about 30 carbon atoms, whichchain optionally includes one or more (e.g., 1, 2, 3, or 4) double ortriple bonds.

In another embodiment, the linker is a divalent branched or unbranchedcarbon chain comprising from about 2 to about 30 carbon atoms.

In another embodiment, the linker is a divalent branched or unbranchedcarbon chain comprising from about 2 to about 20 carbon atoms, whichchain optionally includes one or more (e.g., 1, 2, 3, or 4) double ortriple bonds, and which chain is optionally substituted with one or more(e.g., 2, 3, or 4) hydroxy or oxo (═O) groups.

In another embodiment, the linker is a divalent branched or unbranchedcarbon chain comprising from about 2 to about 20 carbon atoms, whichchain optionally includes one or more (e.g., 1, 2, 3, or 4) double ortriple bonds.

In another embodiment, the linker is a divalent branched or unbranchedcarbon chain comprising from about 2 to about 20 carbon atoms.

In another embodiment, the linker is —(CH₂CH₂O)—₁₋₁₀.

In another embodiment, the linker is —C(═O)NH(CH₂)₃—;—C(═O)NH(CH₂)₅C(═O)NH(CH₂)—; —CH₂OC(═O)NH(CH₂)₂O(CH₂)₂O(CH₂)—;—C(═O)NH(CH₂)₂O(CH₂)₂O(CH₂)₃—; —CH₂C(═O)NH(CH₂)₂O(CH₂)₂O(CH₂)₃—;—(CH₂)₄C(═O)NH(CH₂)₂O(CH₂)₂O(CH₂)₃—;—C(═O)NH(CH₂)₅C(═O)NH(CH₂)₂O(CH₂)₂O(CH₂)₃—.

In another embodiment, the linker comprises one or more divalentheteroaryl groups.

Specifically, (C₁-C₃₀)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, heptyl, octyl,nonyl, or decyl; (C₃-C₈)cycloalkyl can be cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl; (C₂-C₃₀)alkenyl can be vinyl, allyl,1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl,2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl,4-hexenyl, 5-hexenyl, heptenyl, octenyl, nonenyl, or decenyl;(C₂-C₃₀)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl,1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, heptynyl,octynyl, nonynyl, or decynyl; (C₆-C₁₀)aryl can be phenyl, indenyl, ornaphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl,oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl,pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl(or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (orits N-oxide).

The term aromatic includes aryl and heteroaryl groups.

Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclicradical having about nine to ten ring atoms in which at least one ringis aromatic.

Heteroaryl encompasses a radical attached via a ring carbon of amonocyclic aromatic ring containing five or six ring atoms consisting ofcarbon and one to four heteroatoms each selected from the groupconsisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absentor is H, O, (C₁-C₄)alkyl, phenyl or benzyl, as well as a radical of anortho-fused bicyclic heterocycle of about eight to ten ring atomsderived therefrom, particularly a benz-derivative or one derived byfusing a propylene, trimethylene, or tetramethylene diradical thereto.

The term “amino acid,” when used with reference to a linker, comprisesthe residues of the natural amino acids (e.g., Ala, Arg, Asn, Asp, Cys,Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids(e.g., phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline,gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylicacid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,penicillamine, ornithine, citruline, α-methyl-alanine,para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine,and tert-butylglycine). The term also includes natural and unnaturalamino acids bearing a conventional amino protecting group (e.g., acetylor benzyloxycarbonyl), as well as natural and unnatural amino acidsprotected at the carboxy terminus (e.g. as a (C₁-C₆)alkyl, phenyl orbenzyl ester or amide). Other suitable amino and carboxy protectinggroups are known to those skilled in the art (see for example, Greene,Protecting Groups In Organic Synthesis; Wiley: New York, 1981, andreferences cited therein). An amino acid can be linked to anothermolecule through the carboxy terminus, the amino terminus, or throughany other convenient point of attachment, such as, for example, throughthe sulfur of cysteine.

The term “peptide” when used with reference to a linker, describes asequence of 2 to 25 amino acids (e.g. as defined hereinabove) orpeptidyl residues. The sequence may be linear or cyclic. For example, acyclic peptide can be prepared or may result from the formation ofdisulfide bridges between two cysteine residues in a sequence. A peptidecan be linked to another molecule through the carboxy terminus, theamino terminus, or through any other convenient point of attachment,such as, for example, through the sulfur of a cysteine. Preferably apeptide comprises 3 to 25, or 5 to 21 amino acids. Peptide derivativescan be prepared as disclosed in U.S. Pat. Nos. 4,612,302; 4,853,371; and4,684,620. Peptide sequences specifically recited herein are writtenwith the amino terminus on the left and the carboxy terminus on theright.

In one embodiment, a substrate of the invention for a dehalogenase whichhas a linker has the formula (I):R-linker-A-X  (I)wherein R is one or more functional groups (such as a fluorophore,biotin, luminophore, or a fluorogenic or luminogenic molecule, or is asolid support, including microspheres, membranes, polymeric plates,glass beads, glass slides, and the like), wherein the linker is amultiatom straight or branched chain including C, N, S, or O, whereinA-X is a substrate for a dehalogenase, and wherein X is a halogen. Inone embodiment, A-X is a haloaliphatic or haloaromatic substrate for adehalogenase. In one embodiment, the linker is a divalent branched orunbranched carbon chain comprising from about 12 to about 30 carbonatoms, which chain optionally includes one or more (e.g., 1, 2, 3, or 4)double or triple bonds, and which chain is optionally substituted withone or more (e.g., 2, 3, or 4) hydroxy or oxo (═O) groups, wherein oneor more (e.g., 1, 2, 3, or 4) of the carbon atoms in the chain isoptionally replaced with a non-peroxide —O—, —S— or —NH—. In oneembodiment, the linker comprises 3 to 30 atoms, e.g., 11 to 30 atoms. Inone embodiment, the linker comprises (CH₂CH₂O)_(y) and y=2 to 8. In oneembodiment, A is (CH₂)_(n) and n=2 to 10, e.g., 4 to 10. In oneembodiment, A is CH₂CH₂ or CH₂CH₂CH₂. In another embodiment, A comprisesan aryl or heteroaryl group. In one embodiment, a linker in a substratefor a dehalogenase such as a Rhodococcus dehalogenase, is a multiatomstraight or branched chain including C, N, S, or O, and preferably 11-30atoms when the functional group R includes an aromatic ring system or isa solid support.

In another embodiment, a substrate of the invention for a dehalogenasewhich has a linker has formula (II):R-linker-CH₂—CH₂—CH₂—X  (II)where X is a halogen, preferably chloride. In one embodiment, R is oneor more functional groups, such as a fluorophore, biotin, luminophore,or a fluorogenic or luminogenic molecule, or is a solid support,including microspheres, membranes, glass beads, and the like. When R isa radiolabel, or a small detectable atom such as a spectroscopicallyactive isotope, the linker can be 0-30 atoms.

V. Syntheses for Exemplary Substrates[2-(2-Hydroxy-ethoxy)-ethyl]-carbamic acid anthracen-9-ylmethyl ester

To a stirring slurry of 9-anthracenemethanol (10 g, 48 mmol) and4-nitrophenyl chloroformate (13.6 g, 67.5 mmol) in 200 ml CH₂Cl₂ wasadded triethylamine (6.7 ml, 0.19 mol). The resulting gold coloredsolution was allowed to stir 16 hrs at room temperature. At this point,2-(2-aminoethoxy)ethanol (14.4 ml, 0.144 mol) was added and stirringcontinued for another 24 hours. The CH₂Cl₂ reaction mixture was thenwashed with a 2% sodium hydroxide (w/w) solution until no p-nitrophenolwas observed in the organic layer. The dichloromethane was dried withsodium sulfate, filtered, and evaporated under reduced pressure.

The crude product was further purified by column chromatography onsilica gel 60, progressively eluting with 1% to 3% methanol indichloromethane. 7.6 g (58% yield) of a yellow solid was isolated: ¹HNMR (CDCl₃) δ 8.38 (s, H-10), 8.28 (d, H-1, 8), 7.94 (d, H-4, 5), 7.44(m, H-2,3,6,7), 6.06 (s, CH ₂-anth), 5.47 (t, exchangeable, NH), 3.53(bs, CH ₂—OH) 3.33 (m, three —CH ₂—). Mass spectrum, m/e Calcd forC₂₀H₂₂NO₄ ⁺: 340.15. Found: 340.23. Calcd for C₂₀H₂₁NNaO₄ ⁺: 340.15.Found: 340.23.

{2-[2-(6-Chloro-hexyloxy)-ethoxy]ethyl}-carbamic acidanthracen-9-ylmethyl ester

A 100 ml round bottom flask was charged with[2-(2-Hydroxy-ethoxy)-ethyl]-carbamic acid anthracen-9-ylmethyl ester(1.12 g, 3 mmol) and fresh sodium hydride, 60% dispersion in mineral oil(360 mg, 9 mmol) under inert atmosphere. 20 ml anhydrous THF was addedand the reaction allowed to stir for 30 minutes. The flask is thencooled to between −10 and −20° C. by means of an ice/NaCl bath. When thetemperature is reached 1-chloro-6-Iodohexane (1 ml, 6 mmol) is added viasyringe. The reaction is maintained at ice/NaCl temperature for 2 hours,then slowly allowed to warm to room temperature overnight. At this pointsilica gel 60 is co-absorbed onto the reaction mixture with loss ofsolvent under reduced pressure. Silica gel chromatography takes placeinitially with heptane as eluent, followed by 10%, 20%, and 25% ethylacetate. A total of 0.57 g (41% yield) of product is isolated fromappropriate fractions: ¹H NMR (CDCl₃) δ 8.48 (s, H-10), 8.38 (d, H-1,8), 8.01 (d, H-4, 5), 7.52 (dt, H-2,3,6,7), 6.13 (s, CH ₂-anth), 5.29(bs, exchangeable, NH), 3.74 (m, 4H), 3.55-3.15 (m, 8H), 1.84 (m, 4H),1.61 (m, 1H), 1.43 (m, 1H), 1.25 (m, 2H). Mass spectrum, m/e Calcd forC₂₆H₃₂ClNO₄.H₂O: 475.21 (100%), 476.22 (29.6%). Found: 475.21, 476.52.

2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl-ammonium trifluoro-acetate

To {2-[2-(6-Chloro-hexyloxy)-ethoxy]-ethyl}-carbamic acidanthracen-9-ylmethyl ester (0.56 g, 1.2 mmol) dissolved in 4 mldichloromethane was added 2 drops of anisole. The reaction mixture iscooled by means of an ice/NaCl bath. After 10 minutes trifluoroaceticacid (2 ml) is added. The reaction mixture turns dark brown uponaddition and is allowed to stir for 30 minutes. All volatiles areremoved under reduced atmosphere. The residue is re-dissolved in CH₂Cl₂and washed twice with water. The aqueous fractions are frozen andlyophilized overnight. An oily residue remains and is dissolved inanhydrous DMF to be used as a stock solution in further reactions. Massspectrum, m/e Calcd for C₁₀H₂₃ClNO₂ ⁺: 224.14 (100%), 226.14 (32%).Found: 224.2, 226.2.

General methodology for reporter group conjugation to2-[2-(6-chloro-hexyloxy)-ethoxy]-ethylamine

To one equivalent of the succinimidyl ester of the reporter group in DMFis added 3 equivalence of 2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl-ammoniumtrifluoro-acetate stock solution, followed by diisopropylethylamine. Thereaction is stirred from 8 to 16 hours at room temperature. Purificationis accomplished by preparative scale HPLC or silica gel chromatography.

N-{2-[2-(6-Chlorohexyloxy)-ethoxy]-ethyl}-fluorescein-5-amide

The title compound was prepared using the above methodology.Purification was accomplished using preparative scale HPLC. Massspectrum, m/e Calcd for C₃₁H₃₁ClNO₈ ⁻: 580.17 (100%), 581.18 (32%).Found: 580.18, 581.31.

N-{2-[2-(6-Chlorohexyloxy)-ethoxy]ethyl}-biotin-amide

The title compound was prepared using the above methodology.Purification was accomplished using silica gel chromatography (2% to 5%methanol in dichloromethane). Mass spectrum, m/e Calcd forC₂₀H₃₇ClN₃O₄S⁺: 450.22 (100%), 452.22 (32%). Found: 449.95, 451.89.

N-{2-[2-(6-Chlorohexyloxy)-ethoxy]-ethyl}-tetramethylrhodamine-5-(and-6)-amide

The title compound was prepared using the above methodology.Purification was accomplished using preparative scale HPLC. Separationof structural isomers was realized. Mass spectrum, m/e Calcd forC₃₅H₄₃ClN₃O₆ ⁺: 636.28 (100%), 637.29 (39.8%), 638.28 (32.4%). Found:636.14, 637.15, 638.14.

N-{2-[2-(6-Chlorohexyloxy)-ethoxy]ethyl}-rhodamine R110-5-(and -6)-amide

The title compound was prepared using the above methodology.Purification was accomplished using preparative scale HPLC. Separationof structural isomers was realized. Mass spectrum, m/e Calcd forC₃₁H₃₅ClN₃O₆ ⁺: 580.2 (100%), 581.2 (35.6%), 582.2 (32.4%). Found:580.4, 581.4, 582.2.

6-({4-[4,4-difluoro-5-(thiophen-2-yl)-4-bora-3a-4-a-diaza-s-indacene-3-yl]phenoxy}-acetylamino)-hexanoicacid {2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl}-amide

The title compound was prepared using the above methodology.Purification was accomplished using silica gel chromatography (3% to 5%methanol in dichloromethane). Mass spectrum, m/e Calcd forC₃₇H₄₇BClF₂N₄O₅S⁺: 743.3 (100%). Found: 743.4.

6-({4-[4,4-difluoro-5-(thiophen-2-yl)-4-bora-3a-4-a-diaza-s-indacene-3-yl]styryloxy}-acetylamino)-hexanoicacid {2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl}-amide

The title compound was prepared using the above methodology.Purification was accomplished using silica gel chromatography (3%methanol in dichloromethane). Mass spectrum, m/e Calcd forC₃₉H₄₈BClF₂N₄NaO₅S⁺: 791.3 (100%). Found: 7.91.3.

Triethylammonium3-[5-[2-(4-tert-Butyl-7-diethylamino-chromen-2-ylidene)-ethylidene]-3-(5-{2-[2-(6-chlorohexyloxy)-ethoxy]ethylcarbamoyl}-pentyl)-2,4,6-trioxo-tetrahydro-pyrimidin-1-yl]-propane-1-sulfonicacid anion

The title compound was prepared using the above methodology.Purification was accomplished using preparative scale HPLC. Massspectrum, m/e Calcd for C₄₂H₆₂ClN₄O₁₀S⁻: 849.4 (100%), 850.4 (48.8%),851.4 (36.4%). Found: 849.6, 850.5, 851.5.

2-tert-Butyl-4-{3-[1-(5-[2-[2-(6-chlorohexyloxy)-ethoxy]-ethylcarbamoyl}-pentyl)-3,3-dimethyl-5-sulfo-1,3-dihydro-indol-2-ylidene]-propenyl]-7-diethylamino-chromenyliumchloride

The title compound was prepared using the above methodology.Purification was accomplished using preparative scale HPLC. Massspectrum, m/e Calcd for C₄₆H₆₇ClN₃O₇S⁻: 840.4 (100%), 841.4 (54.4%).Found: 840.5, 841.5.

N-{2-[2-(6-Chlorohexyloxy)-ethoxy]-ethyl}-3-{4-[5-(4-dimethylamino-phenyl)-oxazol-2-yl]-benzenesulfonylamino}-propionamide

The title compound was prepared using the above methodology.Purification was accomplished using preparative scale HPLC. Massspectrum, m/e Calcd for C₃₀H₄₀ClN₄O₆S⁻: 619.2 (100%), 620.2 (35%).Found: 619.5, 620.7.

N-{2-[2-(6-Chlorohexyloxy)-ethoxy]-ethyl}-9′-chloroseminaphthofluorescein-5-(and-6)-amide

The title compound was prepared using the above methodology.Purification was accomplished using preparative scale HPLC. Separationof structural isomers was realized. Mass spectrum, m/e Calcd forC₃₅H₃₄Cl₂NO₈ ⁺: 666.17 (100%), 668.16 (64%), 667.17 (39.8%). Found:666.46, 668.44, 667.51.

N-{2-[2-(6-Chlorohexyloxy)-ethoxy]-ethyl}-seminaphthodimethylrhodamine-5-(and-6)-amide

The title compound was prepared using the above methodology.Purification was accomplished using preparative scale HPLC. Massspectrum, m/e Calcd for C₃₇H₃₈ClN₂O₇ ⁻: 657.24 (100%), 658.24 (42%),659.23 (32%). Found: 657.46, 658.47, 659.45.

6-(3′,6′-dipivaloylfluorescein-5-(and -6)-carboxamido) hexanoic acid{2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl}-amide

To a 100 ml round bottom flask containing6-(3′,6′-dipivaloylfluorescein-5-(and -6)-carboxamido) hexanoic acidsuccinimidyl ester (0.195 g, 0.26 mmol) was added2-[2-(6-chlorohexyloxy)-ethoxy]-ethylamine (˜0.44 mmol) in 25 ml Et₂O,followed by 2 ml of pyridine. The reaction mixture was allowed to stirovernight. After evaporation under reduced pressure, the residue wassubjected to silica gel 60 column chromatography, progressively using 2%to 5% methanol in dichloromethane as eluent. The appropriate fractionswere collected and dried under vacuum (0.186 g, 0.216 mmol, and 84%yield). Mass spectrum, m/e Calcd for C₄₇H₆₀ClN₂O₁₁ ⁺: 863.39 (100%),864.39 (54.4%), 865.39 (34.6%). Found: 862.94, 864.07, 864.94.

6-(fluorescein-5-(and -6)-carboxamido) hexanoic acid{2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl}-amide

6-(3′,6′-dipivaloylfluorescein-5-(and -6)-carboxamido) hexanoic acid{2-[2-(6-chlorohexyloxy)-ethoxy]-ethyl}-amide (0.186 g, 0.216 mmol) wasdissolved in 5 ml methanol and 0.5 ml 2M sodium carbonate (aq) added.The reaction mixture was stirred for 16 hours, then filtered.Purification was accomplished using preparative scale HPLC. Separationof structural isomers was realized. Mass spectrum, m/e Calcd forC₃₇H₄₄ClN₂O₉ ⁺: 695.27 (100.0%), 696.28 (42.2%), 697.27 (32.3%). Found:

{2-[2-(4-Chlorobutoxy)-ethoxy]ethyl}-carbamic acid anthracen-9-ylmethylester

A 50 ml round bottom flask was charged with[2-(2-Hydroxyethoxy)-ethyl]-carbamic acid anthracen-9-ylmethyl ester(0.25 g, 0.74 mmol) and fresh sodium hydride, 60% dispersion in mineraloil (150 mg, 3.75 mmol) under inert atmosphere. 10 ml anhydrous THF wasadded and the reaction allowed to stir for 5 minutes. After this point,1-chloro-4-Iodobutane (180 μl, 1.5 mmol) is added via syringe. Thereaction is stirred at room temperature for 24 hours. Silica gel 60 isco-absorbed onto the reaction mixture with loss of solvent under reducedpressure. Silica gel column chromatography takes place initially withheptane as eluent, followed by 10%, 20%, and 30% ethyl acetate. A totalof 0.1 g (32% yield) of product is isolated from appropriate fractions:¹H NMR (CDCl₃) δ 8.50 (s, H-10), 8.40 (d, H-1, 8), 8.03 (d, H-4, 5),7.53 (dt, H-2,3,6,7), 6.15 (s, CH ₂-anth), 5.19 (m, exchangeable, NH),3.93-3.32 (m, 12H) 1.69-1.25 (m, 4H). Mass spectrum, m/e Calcd forC₂₄H₂₈ClNO₄.H₂O: 447.18 (100.0%), 448.18 (27.1%). Found: 447.17, 448.41.

2-(2-{2-[2-(2-Chloroethoxy)-ethoxy]-ethoxy}-ethyl)-isoindole-1,3-dione

2-(2-{2-[2-(2-Hydroxy-ethoxy)-ethoxy]-ethoxy}-ethyl)-isoindole-1,3-dione(0.5 g, 1.55 mmol) was prepared by the method of Nielsen, J. and Janda,K. D. (Methods: A Companion to Methods in Enzymology 6, 361-371 (1994)).To this reagent was added polystyrene-supported triphenylphosphine about3 mmol P/g (0.67 g, 2 mmol) and 6 ml carbon tetrachloride, into a 25 mlround bottom fitted with a reflux condenser. The reaction set-up wassparged with argon then heated to reflux for 2 hours. Upon cooling, morepolystyrene-supported triphenylphosphine (0.1 g, 0.3 mmol) was added andthe reaction refluxed for an additional one hour. The cooled solutionwas filtered and the resin washed with additional carbon tetrachloride.Evaporation of solvent yielded 0.4 g (75.5% yield) of pure titlecompound: ¹H NMR (CDCl₃) δ 7.82 (dd, 2H), 7.69 (dd, 2H), 3.88 (t, 2H),3.71 (q, 4H), 3.63-3.56 (m, 12H). Mass spectrum, m/e Calcd forC₁₆H₂₁ClNO₅ ⁺: 342.11 (100.0%), 344.11 (32.0%). Found: 341.65, 343.64.

2-[2-(2-{2-[2-(2-Chloroethoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl]-isoindole-1,3-dione

The title compound was prepared according to the previous example in 89%yield: ¹H NMR (CDCl₃) δ 7.77 (dd, 2H), δ 7.64 (dd, 2H), 3.83 (t, 2H),3.67 (m, 4H), 3.60-3.52 (m, 14H). Mass spectrum, m/e Calcd forC₁₈H₂₅ClNO₆ ⁺: 386.14 (100.0%), 388.13 (32.0%). Found: 385.88, 387.83.

2-{2-[2-(2-{2-[2-(2-Chloroethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethyl}-isoindole-1,3-dione

The title compound was prepared according to the synthesis of2-(2-{2-[2-(2-Chloro-ethoxy)-ethoxy]-ethoxy}-ethyl)-isoindole-1,3-dionein 92% yield: ¹H NMR (CDCl₃) δ 7.84 (dd, 2H), 7.71 (dd, 2H), 3.90 (t,2H), 3.74 (q, 4H), 3.67-3.58 (m, 18H). Mass spectrum, m/e Calcd forC₂₀H₂₉ClNO₇ ⁺: 430.16 (100.0%). Found: 429.85.

The intermediate compound2-{2-[4-(2-chloroethyl)phenoxy]-ethoxy}ethanaminium chloride, which canbe used to prepare substrates of the invention can be prepared asillustrated below and as described in the following steps a-c.

a. tert-butyl 2-{2-[4-(2-hydroxyethyl)phenoxy]ethoxy}-ethylcarbamate

A 100 ml round bottom flask was charged with 4-hydroxyphenethyl alcohol(1.14 g, 8.2 mmol), cesium carbonate (4.02 g, 12.4 mmol), and tert-butyl2-(2-{[(4-methylphenoxy)sulfonyl]oxy}ethoxy)-ethylcarbamate (2.96 g, 8.2mmol) (prepared using standard chemistry). This reaction mixture wasslurried with 10 ml of DMF and heated to 60° C. by use of an oil bath.The reaction proceeded for 19 hours at which point was it was cooled andthe DMF removed under reduced pressure. Upon adding dichloromethane thereaction mixture was filtered through a plug of celite and then thesolvent removed. The resultant solid was dried under high vacuum. A nearquantitative yield of product was isolated: ¹H NMR (CDCl₃) δ 7.11 (d,2H, Ar), 6.98 (d, 2H, Ar), 4.97 (bs, exchangeable, NH), 4.07 (dd, CH₂—O), 3.79 (t, CH ₂—OH), 3.78 (dd, CH ₂—O), 3.57 (t, CH ₂—O), 3.30 (bm,CH ₂—NH), 2.78 (t, CH ₂—Ar), 1.82 (bs, exchangeable, OH) 1.41 (s, 9H, CH₃). Mass spectrum, m/e Calcd for C₁₇H₂₈NO₅ ⁺: 326.20 (100%), 327.20(19.5%). Found: 326.56, 327.57.

b. tert-butyl 2-{2-[4-(2-chloroethyl)phenoxy]ethoxy}ethylcarbamate

To tert-butyl 2-{2-[4-(2-hydroxyethyl)phenoxy]ethoxy}ethylcarbamate(0.56 g, 1.7 mmol) dissolved in 10 ml carbon tetrachloride was addedtriphenylphosphine bound on styrene (861 mg, 2.6 mmol of about 3 mmol/gresin). The reaction mixture was heated to reflux for 2 hours. After therequired time the reaction was cooled and filtered. After drying aquantitative yield of product was isolated. ¹H NMR (CDCl₃) δ 7.11 (d,2H, Ar), 6.86 (d, 2H, Ar), 4.95 (bs, exchangeable, NH), 4.08 (dd, CH₂—O), 3.79 (dd, CH ₂—O), 3.65 (t, CH ₂—Cl), 3.59 (t, CH ₂—O), 3.32 (bm,CH ₂—NH), 2.99 (t, CH ₂—Ar), 1.70 (bs, exchangeable, OH) 1.42 (s, 9H, CH₃). Mass spectrum, m/e Calcd for C₁₇H₂₇ClNO₄ ⁺: 344.16 (100%), 346.16(32%). Found: 344.57, 346.55.

c. 2-{2-[4-(2-chloroethyl)phenoxy]ethoxy}ethanaminium chloride

tert-butyl 2-{2-[4-(2-chloroethyl)phenoxy]ethoxy}ethylcarbamate (1.7mmol) was dissolved in 5 ml dichloromethane and triethylsilane (0.5 ml,5% v/v) was added. At this point trifluoroacetic acid (5 ml) was addeddropwise to the solution at room temperature. The reaction mixtureturned golden brown upon addition and was allowed to stir for one hour.All volatiles were removed under reduced atmosphere, the residue wasre-dissolved in CH₂Cl₂, and washed twice with dilute HCl. The aqueousfractions were lyophilized overnight. The remaining oily residue wasdissolved in anhydrous DMF to be used as a stock solution in furtherreactions.

The intermediate compound2-(2-{[5-(3-chloropropyl)-2-furyl]methoxy}ethoxy)ethanamine, which canbe used to prepare substrates of the invention can be prepared asillustrated below and as described in the following steps d-g.

d. 2-(t-butyldimethylsilyloxymethyl)furan

To a 1 liter flask containing dimethyl formamide (150 mL) was addedfurfuryl alcohol (17.7 mL, 0.20 mol), t-butyldimethylsilyl chloride(33.7 g, 0.22 mole), and imidazole (15.3 g, 0.22 mol). After 22 hoursstirring at RT, the reaction was filtered and the volatiles removed invacuo. The resulting material was partitioned between diethyl ether (500mL) and a saturated aqueous solution of citric acid (100 mL).Additionally, the ether layer was washed 2×100 mL sat. citric acid. Thecombined aqueous layers were back extracted 1×50 mL ether. The combinedorganic layers were washed 1×100 mL water followed by 1×100 mL brine.The ether layer was dried over anhydrous sodium sulfate, filtered, andevaporated to yield 28.1 g (65% yield). ¹H NMR: (DMSO-d₆) δ 0.01 (s,6H), 0.82 (s, 9H), 4.55 (s, 2H), 6.29 (d, 1H), 6.36 (dd, 1H), 7.58 (d,1H)

e. 2-(3-chloropropyl)-5-(t-butyldimethylsilyloxymethyl)furan

A solution of 2-(t-butyldimethylsilyloxymethyl)furan (5 g, 0.023 mol) inTHF (48 mL) was dried over 3 Å molecular sieves. After the sieves wereremoved, an additional 10 mL THF was added along with TMEDA (3.47 mL,0.023 mol), and the solution was cooled to 0° C. in an ice bath. Asolution of BuLi (10.1 mL of 2.5M in hexane, 0.025 mol) was addeddropwise over 25 minutes. The mixture was allowed to stir for 1 hour.1-chloro-3-iodopropane (5.64 g, 0.028 mol) was injected rapidly. After 2hours TLC indicated completion. The solvent was evaporated, and thematerial was partitioned between ether (100 mL) and 5% citric acid (100mL). The ether layer was washed with water (50 mL) and then brine (50mL). The ether solution was dried with sodium sulfate, filtered, andevaporated. The resulting material was flashed on silica using 20/1heptane/EtOAc. Appropriate fraction were combined and evaporated toyield 4.9 g (75% yield). TLC: R_(f) 0.6 (Heptane/EtOAc 5/1) ¹H NMR:(CDCl₃) δ 0.00 (s, 6H), 0.83 (s, 9H), 2.01 (p, 2H), 2.71 (t, 2H), 3.48(t, 2H), 4.51 (s, 2H), 5.88 (d, 1H), 6.04 (d, 1H)

f. [5-(3-chloropropyl)-2-furyl]methanol

A solution of 2-(3-chloropropyl)-5-(t-butyldimethylsilyloxymethyl)furan(4.88 g, 0.017 mol) in THF (50 mL) was cooled to 0° C. To the abovesolution was added a chilled solution of tetrabutylammonium fluoride (1M in THF, 18.2 mL, 0.018 mol). After 20 minutes, TLC indicated reactioncompletion. Acetic acid (2 mL) was added to the solution. The solutionwas evaporated. The resulting syrup was partitioned between ether (150mL) and sat. citric acid (100 mL). Additionally, the ether layer waswashed with saturated bicarbonate (60 mL and then water (60 mL). Thecombined aqueous layers were back extracted with ether (50 mL). Thecombined ether layers were dried with sodium sulfate, filtered, andevaporated to yield 3.4 g yellow syrup (99% crude yield). The materialwas further purified on silica, eluting with heptane/EtOAc (5/1) toyield 1.6 g (47% yield). TLC: R_(f) 0.5 (Heptane/EtOAc 1/1) ¹H NMR:(CDCl₃) δ 2.05 (p, 2H), 2.74 (t, 2H), 3.21 (bs, 1H), 3.51 (t, 2H), 4.45(s, 2H), 5.94 (d, 1H), 6.11 (d, 1H)

g. 2-(2-{[5-(3-chloropropyl)-2-furyl]methoxy}ethoxy)ethanamine

A solution of [5-(3-chloropropyl)-2-furyl]methanol (500 mg, 2.5 mmol) inether (6 mL) was dried over 3 {acute over (Å)} molecular sieves. Afterthe sieves were removed, pyridine (245 μL, 3.0 mmol) was added to thesolution. Thionyl bromide (215 μL, 2.9 mmol) was added dropwise to thesolution. After 7 hours of stirring, the solution was rapidly injectedinto a solution of sodium 2-[2-aminoethoxy]ethoxide (534 mg, 4.2 mmol)in DMF (3 mL). (Sodium 2-[2-aminoethoxy]ethoxide was previously preparedby adding 60% NaH dispersion (2.85 g, 0.07 mol) subsequently cleanedwith heptane to a solution of 2-aminoethoxyethanol (5 g, 0.04 mol) indiglyme (10 mL), stirred for 5 hours, and evaporated.) After 2 hours thereaction was placed in the freezer. After 18 hours the reaction waspartitioned between dichloromethane (DCM) (50 mL) and water (50 mL).Water layer was extracted with additional 30 mL DCM. The combined DCMlayers were washed with water (30 mL). The DCM layer were extracted withdiluted HCl (1N, 30 mL) followed by water (20 mL). The acidic aqueoussolutions were adjusted to pH=10 with diluted sodium hydroxide and backextracted with 2×20 ml DCM. The DCM was washed with brine, dried withsodium sulfate, filtered, and evaporated to yield 380 mg (58% yield).Used without further purification. TLC: R_(f) 0.5 (IPA/NH₄OH/water 8/1/1exposed with ninhydrin solution) Mass spectrum, m/e Calcd forC₁₂H₂₁ClNO₃ ⁺: 262.1 (100%), 264.1 (32%). Found: 262.6, 264.6.

General methodology for reporter group conjugation to2-{2-[4-(2-chloroethyl)phenoxy]ethoxy}ethanamine or2-(2-{[5-(3-chloropropyl)-2-furyl]methoxy}ethoxy)ethanamine

To one equivalent of the succinimidyl ester of the reporter group in DMFis added 1.5 equivalents of substrate stock solution, followed bydiisopropylethylamine. The reaction is stirred from 8 to 16 hours atroom temperature. Purification is accomplished by preparative scale HPLCor silica gel chromatography.

Using the General Procedure above, the following substrates (XXIX-XXXIV)of the invention were prepared.

2-{2-[4-(2-chloroethyl)phenoxy]ethoxy}ethyl-tetramethylrhodamine-6-carboxamide

The title compound was prepared using the general methodology startingwith 6-carboxytetramethylrhodamine succinimidyl ester. Purification wasaccomplished using preparative scale HPLC. Separation of structuralisomers was realized. UV/Vis (MeOH): 544(max) Mass spectrum, m/e Calcdfor C₃₇H₃₉ClN₃O₆ ⁺: 656.25 (100%), 658.25 (32.4%). Found: 656.37,658.37. This compound is referred to herein ascarboxytetramethylrhodamine-p-phenethyl-Cl.

2-{2-[4-(2-chloroethyl)phenoxy]ethoxy}ethyl-fluorescein-5-(and-6)-carboxamide

The title compounds were prepared using the general methodology startingwith 5(6)-carboxyfluorescein succinimidyl ester. Purification andseparation of isomers was accomplished using preparative scale HPLC.Mass spectrum, m/e Calcd for C₃₃H₂₇ClNO₈ ⁻: 600.14 (100%), 601.15(37.4%), 602.14 (32.1%). Found: 600.18, 601.24, 602.21. This compound isreferred to herein as carboxyfluorescein-p-phenethyl-Cl.

2-{2-[4-(2-chloroethyl)phenoxy]ethoxy}ethyl-biotin-carboxamide

The title compound was prepared using the general methodology startingwith D-biotin succinimidyl ester. Purification was accomplished usingpreparative scale HPLC. Mass spectrum, m/e Calcd for C₂₂H₃₃ClN₃O₄S⁺:470.19 (100%). Found: 470.19. This compound is referred to herein asbiotin-p-phenethyl-14-Cl.

2-{2-[4-(2-chloroethyl)phenoxy]ethoxy}ethyl-3′,6′-diacetylfluorescein-6-carboxamide

To a 10 ml round bottom flask containing eitherN-{2-[4-(2-chloroethyl)-1-ethoxyphenyl]ethyl}-fluorescein-6-carboxamide(12.3 mg,) was added 2 ml of acetic anhydride followed by 0.25 ml ofpyridine. The reaction mixture was allowed to 1 hour. After evaporationunder reduced pressure, the residue was co-evaporated with toluene twotimes. The solid was then dried under vacuum (0.186 g, 0.216 mmol, and84% yield). Mass spectrum, m/e Calcd for C₃₇H₃₃ClNO₁₀ ⁺: 686.18 (100%),687.18 (41.9%), 688.18 (34.1%). Found: 686.55, 687.61, 688.60.

N-[2-(2-{[5-(3-chloropropyl)-2-furyl]methoxy}ethoxy)ethyl]tetramethylrhodamine-6-carboxyamide

The title compound was prepared using the general methodology startingwith 6-carboxytetramethylrhodamine succinimidyl ester. Purification wasaccomplished using preparative scale HPLC. ¹H NMR: (CD₃OD) δ 2.04 (p,2H), 2.75 (t, 2H), 3.26 (s, 12H), 3.60 (m, 10H), 4.38 (s, 2H), 5.97 (d,1H), 6.20 (d, 1H) 6.99 (d, 2H), 7.08 (dd, 2H), 7.15 (d, 2H), 7.81 (s,1H), 8.19 (d, 1H), 8.39 (d, 1H), 8.73 (bt, 1H) Mass spectrum, m/e Calcdfor C₃₇H₄₁ClN₃O₇ ⁺: 674.26 (100.0%), 675.27 (42.0%), 676.26 (32.4%)Found: 674.5, 675.5, 676.5. This compound is referred to herein ascarboxytetramethylrhodamine-furanyl-propyl-Cl.

N-[2-(2-{[5-(3-chloropropyl)-2-furyl]methoxy}ethoxy)ethyl]-fluorescein-6-carboxamide

The title compounds were prepared using the general methodology startingwith 5(6)-carboxyfluorescein succinimidyl ester. Purification andseparation of isomers was accomplished using preparative scale HPLC.Mass spectrum, m/e Calcd for C₃₃H₃₁ClNO₉ ⁺: 620.17 (100%), 602.17(32.1%). Found: 620.47, 622.49. This compound is referred to herein ascarboxyfluorescein-furanyl-propyl-Cl.

VI. Exemplary Methods of Use

The invention provides methods to monitor the expression, locationand/or trafficking of molecules in a cell, as well as to monitor changesin microenvironments within a cell, and to isolate, image, identify,localize, display or detect one or more molecules which may be presentin a sample, e.g., in a cell, which methods employ a hydrolase substrateand/or a mutant hydrolase of the invention. The substrates of theinvention are preferably soluble in an aqueous or mostly aqueoussolution, including water and aqueous solutions having a pH greater thanor equal to about 6. Stock solutions of substrates of the invention,however, may be dissolved in organic solvent before diluting intoaqueous solution or buffer. Preferred organic solvents are aprotic polarsolvents such as DMSO, DMF, N-methylpyrrolidone, acetone, acetonitrile,dioxane, tetrahydrofuran and other nonhydroxylic, completelywater-miscible solvents. The concentration of a substrate of theinvention and a corresponding mutant hydrolase to be used is dependentupon the experimental conditions and the desired results, e.g., toobtain results within a reasonable time, with minimal background orundesirable labeling. The concentration of a substrate of the inventiontypically ranges from nanomolar to micromolar. The requiredconcentration for the substrate of the invention with a correspondingmutant hydrolase is determined by systematic variation in substrateuntil satisfactory labeling is accomplished. The starting ranges arereadily determined from methods known in the art.

In one embodiment, a substrate which includes a functional group withoptical properties is employed with a mutant hydrolase to label asample. Such a substrate is combined with the sample of interestcomprising the mutant hydrolase for a period of time sufficient for themutant hydrolase to bind the substrate, after which the sample isilluminated at a wavelength selected to elicit the optical response ofthe functional group. Optionally, the sample is washed to removeresidual, excess or unbound substrate. In one embodiment, the labelingis used to determine a specified characteristic of the sample by furthercomparing the optical response with a standard or expected response. Forexample, the mutant hydrolase bound substrate is used to monitorspecific components of the sample with respect to their spatial andtemporal distribution in the sample. Alternatively, the mutant hydrolasebound substrate is employed to determine or detect the presence orquantity of a certain molecule. In another embodiment, the mutanthydrolase bound substrate is used to analyze the sample for the presenceof a molecule that responds specifically to the functional group.

In contrast to intrinsically fluorescent proteins, e.g., GFP, a mutanthydrolase bound to a fluorescent substrate does not require a nativeprotein structure to retain fluorescence. After the fluorescentsubstrate is bound, the mutant hydrolase may be detected, for example,in denaturing electrophoretic gels, e.g., SDS-PAGE, or in cells fixedwith organic solvents, e.g., paraformaldehyde. Fragments of the mutanthydrolase that contain the reactive nucleophilic amino acid may also bedetected by the bound fluorophore, for example, to monitor proteolyticprocesses.

A detectable optical response means a change in, or occurrence of, aparameter in a test system that is capable of being perceived, either bydirect observation or instrumentally. Such detectable responses includethe change in, or appearance of, color, fluorescence, reflectance,chemiluminescence, light polarization, light scattering, or X-rayscattering. Typically the detectable response is a change influorescence, such as a change in the intensity, excitation or emissionwavelength distribution of fluorescence, fluorescence lifetime,fluorescence polarization, or a combination thereof. The detectableoptical response may occur throughout the sample comprising a mutanthydrolase or a fusion thereof or in a localized portion of the samplecomprising a mutant hydrolase or a fusion thereof. Comparison of thedegree of optical response with a standard or expected response can beused to determine whether and to what degree the sample comprising amutant hydrolase or a fusion thereof possesses a given characteristic.

In another embodiment, the functional group is a ligand for an acceptormolecule. Where the substrate comprises a functional group that is amember of a specific binding pair (a ligand), the complementary member(the acceptor) or the substrate, may be immobilized on a solid orsemi-solid surface, such as a polymer, polymeric membrane or polymericparticle (such as a polymeric bead), or both may be in solution. In oneembodiment, protein-protein interactions may be detected using anelectrical conducting substrate coated surface. Representative specificbinding pairs include biotin and avidin (or streptavidin oranti-biotin), IgG and protein A or protein G, drug and drug receptor,toxin and toxin receptor, carbohydrate and lectin or carbohydratereceptor, peptide or protein and peptide or protein receptor, two ormore proteins which interact, for instance, protein kinase A (PKA)regulatory subunit and PKA catalytic subunit, an enzyme and itssubstrate, e.g., a protease, kinase, or luciferase and a substratetherefor, a cofactor for an enzyme and the enzyme, sense DNA or RNA andantisense (complementary) DNA or RNA, hormone and hormone receptor, andion and chelator, and the like. Ligands for which naturally occurringreceptors exist include natural and synthetic proteins, including avidinand streptavidin, antibodies, enzymes, and hormones; nucleotides andnatural or synthetic oligonucleotides, including primers for RNA andsingle- and double-stranded DNA; lipids; polysaccharides andcarbohydrates; and a variety of drugs, including therapeutic drugs anddrugs of abuse and pesticides. Where the functional group is a chelatorof calcium, sodium, magnesium, potassium, or another biologicallyimportant metal ion, the substrate comprising such a functional groupfunctions as an indicator of the ion. Alternatively, such a substratemay act as a pH indicator. Preferably, the detectable optical responseof the ion indicator is a change in fluorescence.

A sample comprising a mutant hydrolase or a fusion thereof is typicallylabeled by passive means, i.e., by incubation with the substrate.However, any method of introducing the substrate into the samplecomprising a mutant hydrolase or a fusion thereof, such asmicroinjection of a substrate into a cell or organelle, can be used tointroduce the substrate into the sample comprising a mutant hydrolase ora fusion thereof. The substrates of the present invention are generallynon-toxic to living cells and other biological components, within theconcentrations of use.

A sample comprising a mutant hydrolase or a fusion thereof can beobserved immediately after contact with a substrate of the invention.The sample comprising a mutant hydrolase or a fusion thereof isoptionally combined with other solutions in the course of labeling,including wash solutions, permeabilization and/or fixation solutions,and other solutions containing additional detection reagents. Washingfollowing contact with the substrate generally improves the detection ofthe optical response due to the decrease in non-specific backgroundafter washing. Satisfactory visualization is possible without washing byusing lower labeling concentrations. A number of fixatives and fixationconditions are known in the art, including formaldehyde,paraformaldehyde, formalin, glutaraldehyde, cold methanol and 3:1methanol:acetic acid. Fixation is typically used to preserve cellularmorphology and to reduce biohazards when working with pathogenicsamples. Selected embodiments of the substrates are well retained incells. Fixation is optionally followed or accompanied bypermeabilization, such as with acetone, ethanol, DMSO or variousdetergents, to allow bulky substrates of the invention, to cross cellmembranes, according to methods generally known in the art. Optionally,the use of a substrate may be combined with the use of an additionaldetection reagent that produces a detectable response due to thepresence of a specific cell component, intracellular substance, orcellular condition, in a sample comprising a mutant hydrolase or afusion thereof. Where the additional detection reagent has spectralproperties that differ from those of the substrate, multi-colorapplications are possible.

At any time after or during contact with the substrate comprising afunctional group with optical properties, the sample comprising a mutanthydrolase or a fusion thereof is illuminated with a wavelength of lightthat results in a detectable optical response, and observed with a meansfor detecting the optical response. While some substrates are detectablecolorimetrically, using ambient light, other substrates are detected bythe fluorescence properties of the parent fluorophore. Uponillumination, such as by an ultraviolet or visible wavelength emissionlamp, an arc lamp, a laser, or even sunlight or ordinary room light, thesubstrates, including substrates bound to the complementary specificbinding pair member, display intense visible absorption as well asfluorescence emission. Selected equipment that is useful forilluminating the substrates of the invention includes, but is notlimited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps,argon lasers, laser diodes, and YAG lasers. These illumination sourcesare optionally integrated into laser scanners, fluorescence microplatereaders, standard or mini fluorometers, or chromatographic detectors.This colorimetric absorbance or fluorescence emission is optionallydetected by visual inspection, or by use of any of the followingdevices: CCD cameras, video cameras, photographic film, laser scanningdevices, fluorometers, photodiodes, quantum counters, epifluorescencemicroscopes, scanning microscopes, flow cytometers, fluorescencemicroplate readers, or by means for amplifying the signal such asphotomultiplier tubes. Where the sample comprising a mutant hydrolase ora fusion thereof is examined using a flow cytometer, a fluorescencemicroscope or a fluorometer, the instrument is optionally used todistinguish and discriminate between the substrate comprising afunctional group which is a fluorophore and a second fluorophore withdetectably different optical properties, typically by distinguishing thefluorescence response of the substrate from that of the secondfluorophore. Where the sample comprising a mutant hydrolase or a fusionthereof is examined using a flow cytometer, examination of the samplecomprising a mutant hydrolase or a fusion thereof optionally includesisolation of particles within the sample comprising a mutant hydrolaseor a fusion thereof based on the fluorescence response of the substrateby using a sorting device.

In one embodiment, a mutant hydrolase and a corresponding substratewhich includes a functional group are employed to label a cell, e.g., acell in an organism such as a cell in transgenic animal, a cell in ananimal administered cells comprising the mutant hydrolase and/orsubstrate of the invention, or cells in culture, or a cellularcomponent. For instance, cells are contacted with a vector encoding themutant hydrolase, such as one encoding a fusion between the mutanthydrolase and a nuclear localization signal. The expression of thevector in the cell which may be in a transgenic animal or administeredto an animal, may be transient, regulatable or stable. Then the cell oran animal comprising the cell is contacted with a substrate of theinvention recognized by the mutant hydrolase. Alternatively, cells areconcurrently contacted with the vector and the substrate. Then thepresence or location of the functional group in the animal, cell, alysate thereof, or a subcellular fraction thereof, is detected ordetermined. In another embodiment, a mutant hydrolase and acorresponding substrate which includes a functional group comprising atriplet sensitizer are employed to selectively inactivate or destroy amolecule and/or cellular activity, e.g., in a cell. In this embodiment,after contacting a sample comprising mutant hydrolase or a fusionthereof with a substrate comprising a triplet sensitizer, the sample isexposed to UV light.

To label proteins in vitro or in vivo, a hydrolase substrate may beattached to an amino acid or a tRNA, e.g., an aminoacylated tRNA such asan aminoacylated initiator methionyl tRNA for N-terminal modification ofin vitro synthesized proteins, to an amber suppressor tRNA forC-terminal labeling of proteins, including amino acids attached to atRNA using a mutant tRNA synthetase. A hydrolase substrate may also beattached to a protein by an intein-mediated method. The protein ofinterest is expressed as a fusion protein with a carboxyl terminalintein domain, preferably a “mini-intein” lacking a homing endonucleasedomain, and more preferably the Mycobacterium xenopi (Mxe) GyrAmini-intein. Treatment of the fusion protein with a reducing thiolreagent, such as reduced sodium 2-mercaptoethanesulfonate or2-mercaptoethanol, in the presence of the cysteine-hydrolase substrate,e.g., cysteine-haloalkane, results in cleavage of the fusion protein atthe amino-terminal cysteine residue of the intein portion of the fusionand covalent attachment of the cysteine-hydrolase substrate, e.g.,cysteine-haloalkane, to the carboxyl terminus of the protein ofinterest.

Accordingly, proteins can be expressed from cDNA or mRNA without theneed for making fusion proteins and those proteins can be purified usinga mutant hydrolase. Moreover, protein microarrays can be made from thein vitro translated proteins, for instance, using immobilized mutanthydrolase, without the need for a fusion tag, and those proteins as wellas proteins which interact with those proteins, isolated. Further, theuse of a substrate which includes a fluorophore allows for the rapiddetection, as well as the purification, of expressed proteins. For invivo labeling of proteins in a cell, a substrate which includes amethionine or other naturally occurring or nonnaturally occurring aminoacid may be employed, and newly synthesized proteins as well as proteinswhich interact with those proteins, can be purified optionally using animmobilized mutant hydrolase, without the need for a fusion tag. Thisapproach may also be used for isolating marker proteins for differentialprotein expression analysis, and also with mass spectrometry.Multiplexing is also possible using substrates with differentfluorophores.

The substrates and mutant hydrolases of the invention are particularlyuseful to isolate, display or detect molecules in a sample. In oneembodiment, a protein microarray may be prepared in which a mutanthydrolase is immobilized onto a surface of a solid support and asubstrate of the invention modified to include one or more functionalgroups which bind a single protein, a functional or structural class ofproteins, or proteins in general, for protein immobilization (FIG. 57).For example, fusion protein systems such as a thioredoxin patch, inteinbased approaches or other methods are employed to immobilize a mutanthydrolase onto a solid surface. Modified substrates for immobilizingproteins are then added. The substrate may be modified with succinimidylester/aldehyde (for general immobilization of proteins), glutathione(for immobilizing GST fusion proteins), NTA or metal (for immobilizingHis-tagged proteins), or specific ligands for immobilizing specificclasses of proteins. For example, an enzyme substrate or an inhibitor ofan enzyme linked to a hydrolase substrate may be used for immobilizing aparticular class of enzymes, e.g., caspases or reverse transcriptases,or a DNA which binds certain proteins linked to a hydrolase substratemay be used to prepare a protein microarray for DNA binding proteins,e.g., for developing chip based assays. Similarly, to studyprotein-protein interactions including isolating protein complexes, amutant hydrolase can be immobilized on magnetic or non-magneticparticles, e.g., MagneSil particles. These methods can avoid preparingnew fusion proteins, e.g., a new library, as only a substrate for aprotein(s) of interest needs to be prepared, for instance, for GSTfusion libraries.

Alternatively, the substrate may be immobilized onto a surface to allowstable attachment of mutant hydrolases, and fusions thereof, onto thesurface. The mutant hydrolases may be obtained from living cells or bycell-free methods, e.g., coupled transcription and translation in acellular lysate. The bound proteins may be useful for analyzingcharacteristics such as binding to other molecules or enzymaticactivity. It may also be useful for stably immobilizing an enzymaticactivity for bioconversions or detection capabilities. It may also beuseful for stably immobilizing a specific binding activity forpurification or selective adsorption capabilities. Multiple substratesmay be immobilized onto the surface, either at different locations or asa mixture, to allow attachment of multiple mutant hydrolases. Thesubstrate may also be immobilized with other binding molecules such asbiotin or para-substituted benzylguanine.

In one embodiment, a fusion to a mutant hydrolase may be used toidentify proteins that bind to the fusion. Proteins that bind to thefusion protein may be separated from other unbound proteins by bindingthe mutant hydrolase to a substrate immobilized onto a surface. By thismeans, the unbound proteins in solution may be washed from thestationary bound proteins. Other molecules that bind to the fusionprotein, such as nucleic acids or small molecules, may also beidentified by this method. To increase the binding stability of themolecules bound to the mutant hydrolase, various chemical cross-linkingmethods may be employed to covalently interconnect the bound molecules.Reversible cross-linkers are preferred, so that the bound molecules maybe subsequently unbound for analysis, e.g., identification and/orisolation.

After combining a mutant hydrolase with a substrate, it may be necessaryto inactivate remaining unreacted substrate in the mixture. This may bedone by adding wild-type hydrolase to the mixture to convert theremaining unreacted substrate into product. For example, unreactedchloroalkane substrate may be converted to the corresponding alcohol byaddition of a wild-type or other catalytically competent dehalogenase.The unreacted substrate may be free in solution or bound to a surface.If bound to a surface, addition of a catalytically competent hydrolasewould convert the substrate its corresponding product, therebypreventing further binding of mutant hydrolase to the surface.

The substrates and mutant hydrolases of the invention may also be usedin tandem affinity purification (TAP), a method for the purification ofproteins or protein complexes, which uses two consecutive affinitypurification steps. Each purification step employs a ligand for anaffinity tag, for instance, His-tag, a GST-tag, a Strep-tag, abiotin-tag, an immunoglobulin binding domain, e.g., an IgG bindingdomain, a calmodulin binding peptide and the like, which is fused to aprotein of interest. A mutant hydrolase of the invention may be employedas an affinity tag. For example, a fusion containing a mutant hydrolaseand calmodulin binding peptide (CBP) or protein complexes therewith maybe purified by calmodulin attached to a solid phase followed by ahydrolase substrate attached to a solid phase. The purified proteins orcomplexes may then be analyzed by Western blotting or mass spectrometry.Using TAP, proteins or protein complexes may be purified from varioustypes of host cells, such as bacteria, Drosophila, plant, mammaliancells, as well as cell free protein expression systems, and can identifyprotein-protein interactions. The use of a mutant hydrolase andhydrolase substrate in TAP, e.g., for a final affinity purificationstep, allows for the analysis of proteins in real-time, followed by TAPat various time points or after various drug treatments. Since themutant hydrolase fusion is attached covalently to the substrate,purified protein complexes will not contain the hydrolase.

In another embodiment, a biotinylated hydrolase substrate binds avidinlabeled antibodies which bind to an antigen. This complex may besubjected to immunoprecipitation, e.g., by using eppendorff tubescontaining immobilized mutant hydrolase (FIG. 58).

To detect some molecules, a solution (free) or immobilized system may beemployed. In one embodiment, a hydrolase substrate modified with a smallmolecule or a compound could be used for detecting modifications of theattached small molecule or a compound. For example, to detect a kinasesuch as phosphatidylinositol 3 (PI3) kinase, a hydrolase substratemodified with a lipid such as phosphatidylinositol is contacted with asample containing a PI3 kinase, which phosphorylatesphosphatidylinositol. The resulting modified hydrolase substrate is thencovalently attached to the hydrolase. The phosphorylatedphosphatidylinositol is detected by electrophoretic or fluorescencemethods. Electrophoretic detection methods include performing a standardkinase assay using radiolabeled nucleotides such as gamma ³²PATPfollowed by autoradiography or by fluorescence detection usingfluorescently labeled NTA complexed with Ga³⁺ or Fe³⁺. Specific bindingof Ga³⁺ or Fe³⁺ complexed NTA to phosphate groups allows for theelectrophoretic detection of phosphorylated phosphatidylinositol. PI3kinase activity may also be detected in free solution using FRET orfluorescence polarization (FP). For this, a fluorescently labeled,phosphatidylinositol containing hydrolase substrate may be used.Phosphorylated phosphatidylinositol is detected using a differentfluorophore labeled NTA complexed with Ga³⁺ or Fe³⁺. For fluorescencepolarization (FP), a nonfluorescent phosphatidylinositol containinghydrolase substrate is added to a test sample, followed by the additionof Ga³⁺ or Fe³⁺. The resulting hydrolase substrate is added toimmobilized or free mutant hydrolase. Fluorescence polarization isassayed using fluorescence labeled NTA, which binds to Ga³⁺ or Fe³⁺.

To detect phosphodiesterase, a hydrolase substrate which includesfluorescently labeled cAMP and a different fluorophore may be employed.Hydrolysis of cAMP indicates phosphodiesterase activity.Phosphodiesterase activity can be detected using FRET after capturingthe substrate with free or immobilized dehalogenase, e.g., in proteinmicroarray format.

Nucleic acid molecules attached to a hydrolase substrate may be employedto purify or display other nucleic acid molecules, proteins or proteinbased complexes. For ribosome display or purification, e.g., for use indirected evolution, a hydrolase substrate is bound at the 3′ end of amRNA without a stop codon. The substrate is added to an in vitrotranslation mixture and the resulting protein-DNA-mRNA complex ispurified using immobilized mutant hydrolase. Similarly, to isolate,detect or display specific genes, a hydrolase substrate which includesfluorophore labeled DNA or RNA, e.g., a fluorophore labeled singlestranded DNA, which binds a gene of interest, and a differentfluorophore or quencher, is used to isolate, detect or display that genefrom a complex mixture using fluorescence based methods such as FRET.Such a method could be useful in diagnostics as well as bioweapondetection.

The substrates and mutant hydrolases of the invention may be employed invarious formats to detect cAMP (FIGS. 59A-B). In one embodiment,fluorescence quenching is used with two fusion proteins and twosubstrates. One substrate includes a fluorophore and the other includesquencher dye for the fluorophore. One fusion protein includes a mutanthydrolase and the regulatory subunit of PKA, and the other includes amutant hydrolase and PKA catalytic subunit (FIG. 59A). Each fusionprotein is contacted with one of the substrates and then the complexesare mixed together. In presence of cAMP, the quencher dye is no longerin close proximity to the fluorophore. Thus, cAMP is measured bymeasuring fluorescence. In another embodiment, two hydrolase substrateseach with a different fluorophore are employed and cAMP is measured bymeasuring FRET.

In another embodiment, one fusion protein includes a first mutanthydrolase and the regulatory subunit of PKA, and the other fusionprotein includes a protein that is different than the first mutanthydrolase (a second protein) and binds a second substrate and the PKAcatalytic subunit. The mutant hydrolase binds a hydrolase substrate thatincludes at least one fluorophore. The second protein binds a secondsubstrate, which is modified with a quencher for the at least onefluorophore that does not affect the substrate's binding to the secondprotein. The second protein may be GST, thioredoxin, AGT, a differentmutant hydrolase which is specific for a different substrate than thefirst mutant hydrolase, a mutant hydrolase that is capable of bindingthe same substrate as the first mutant hydrolase, or other substratebinding protein. Each fusion protein is contacted with the respectivesubstrate, the complexes are mixed together, and cAMP is measured bymeasuring fluorescence. In the presence of cAMP, the quencher is nolonger in close proximity to the fluorophore. In another embodiment, thesecond protein binds a second substrate which is modified with adifferent fluorophore than the fluorophore linked to the hydrolasesubstrate, which different fluorophore does not affect the binding ofthe second substrate to the second protein. Each fusion protein iscontacted with its respective substrate, the complexes are mixedtogether, and FRET employed to measure cAMP. In the presence of cAMP,the two fluorophores are no longer in close proximity. In yet anotherembodiment, one fusion protein includes a mutant hydrolase and theregulatory subunit of PKA, and the other fusion protein includes afluorescent protein and the PKA catalytic subunit. Fluorescent proteinsinclude but are not limited to GFP, YFP, EGFP, and DsRed. Each fusionprotein is contacted with its respective substrate and then thecomplexes are mixed together. In the presence of cAMP, the fluorescenceprotein and the mutant hydrolase bound to the fluorophore containingsubstrate are no longer in close proximity. In another embodiment, BRETis employed to detect cAMP. One substrate which includes a fluorophoreis contacted with a fusion protein which includes a mutant hydrolase anda regulatory subunit of PKA, and another fusion which includes aluciferase and a regulatory subunit of PKA. When the regulatory subunitfrom each fusion protein dimerizes, BRET is observed. BRET is disruptedin presence of cAMP (see FIG. 59B).

A mutant hydrolase and substrate may be employed in molecularimprinting, a technique devised to generate a polymeric material that isanalyte specific. Molecular imprinting is a process for preparingpolymers that are selective for a particular compound (the printmolecule) (Arshady et al., 1981). The technique involves: (1)prearranging the print molecule and the monomers and allowingcomplementary interactions (non-covalent or reversible covalent) todevelop; (2) polymerizing around the print molecule-monomer complex; and(3) removing the print molecule from the polymer by extraction.Polymerization thus preserves the complementarity to the print moleculeand the polymer will selectively adsorb the print molecule. Molecularlyimprinted polymers (MIPS) with a hydrolase substrate bind to thehydrolase and fusions thereof, and may be used to purify fusionproteins, prepare protein microarrays, study protein-proteininteraction, and TAP.

The functional group of the substrate may bind to another protein,either reversibly or covalently. An example of a functional group thatbinds reversibly to a protein is a hapten that binds to an antibody,e.g., a single-chain antibody (scFv). An example of a functional groupthat binds covalently to another protein is a chloroalkane that binds toa mutant dehalogenase, or a para-substituted benzylguanine that binds toO-alkylguanine-DNA alkyltransferase (AGT). A first fusion proteincomprising a mutant hydrolase may be bound to a second protein as ameans for implementing or modulating a biochemical or biologicalprocess. For example, gene transcription may be modulated by a DNAbinding protein fused to a mutant hydrolase bound to a transcriptionalactivator, e.g., VP16. In such an example, gene transcription would beincreased by addition of a substrate causing the mutant hydrolase fusedto the DNA binding protein to bind to the transcriptional activator. Inanother example, the activity of a protein in a cell may be modulated byits location(s) within the cell. For example, the activity of a proteinmay be changed by binding the protein to a mutant hydrolase fused to asecond protein, which upon binding redirects or preferentiallyredistributes the protein to a different subcellular compartment. Anexample may be a transcription factor located predominately in thenon-nuclear portion of a cell, where upon binding to a mutant hydrolasefused to a nuclear targeting sequence, results in the transcriptionfactor moving to the nucleus. In such an example, the addition of asubstrate to cause binding of the transcription factor to the mutanthydrolase may thereby modulate gene expression mediated by thetranscription factor.

The substrate may have multiple reactive groups to allow interconnectionof mutant hydrolases. When fused to proteins having a binding activity,interconnection of the mutant hydrolases may yield a multivalent bindingcomplex. Such multivalent complexes may have useful properties, such ashigher apparent binding efficiency (e.g., higher avidity). For example,interconnecting two or more single-chain antibodies may yield moreefficient binding to the corresponding antigen. In another example, theDNA binding domain of a lambda phage repressor protein fused to a mutanthydrolase may bind more efficiently to DNA upon addition of a substrateto interconnect the mutant hydrolases. Multivalent complexes havingdifferent binding proteins fused to mutant hydrolases can allowdifferent molecules, e.g., antigens, to be bound together via thecomplex.

Fusing mutant hydrolases together may allow multiple substrates to bindto a single protein. These substrates may be the same or different. Bythis means, the fused mutant hydrolases may serve as a bridging moleculebetween the substrates. This may be useful to covalently interconnectmolecules, such as functional groups, surfaces, or other molecules. Forexample, a substrate bound to a surface may be covalently attached to apolynucleotide bound to a substrate by using a bi-valent fused mutanthydrolase. Hetero-multivalent molecules may be made by fusing differentmutant hydrolases, or fusing mutant hydrolases to other protein(s)capable of making stable covalent bonds, e.g., AGT.

In one embodiment, a substrate includes more than one functional group,e.g., an optically detectable molecule and a ligand for an acceptormolecule, two different proteins, e.g., AGT and a fluorescent protein ora luciferase, an optically detectable molecule and a proteaserecognition site, or an optically detectable molecule and a proteaserecognition site, and a quencher of the optically detectable molecule.For example, a substrate of the invention may include a fluorophore, aprotease recognition site and a quencher molecule. The substrate istaken up by a cell which expresses the mutant hydrolase. In the presenceof the protease, the quencher is removed from the substrate, resultingin a fluorescence signal. The use of such a substrate can yield areal-time assay for the protease. The mutant may also be used for thedetection of infectious agents and thus may be employed in clinicaldiagnostic applications as well as to detect bioweapons.

Other formats may be used to detect proteases such as caspases or aproteosome, e.g., the 20S proteosome may be detected (or isolated) witha branched peptide substrate. In one embodiment, a gene for a mutanthydrolase or another reporter protein, e.g., a luciferase, is used in amammalian cell based expression system. In one embodiment, a protease,e.g., a caspase, recognition site is introduced into a protein which isa transcription repressor protein, e.g., a tet repressor protein or alac repressor protein. In one embodiment, a protease recognition site isintroduced into a loop region of the repressor protein or another regionthat does not inhibit the repressor function for the protein. A vectorwhich includes a promoter linked to DNA which binds the transcriptionrepressor protein linked to the reporter gene is introduced to a cellwhich contains the modified transcription repressor protein. In absenceof the protease, the modified transcription repressor protein inhibitsthe expression of the reporter gene. In the presence of the protease,the modified transcription repressor protein is inactivated due toproteolytic cleavage of the protease site. As a result, the modifiedtranscription repressor protein is not able repress transcription, whichresults in the expression of the reporter gene (FIG. 60A). In oneembodiment, the modified transcription repressor protein gene and/or thereporter gene are stably transfected into cells. Such an assay may beused in conjunction with other assays, including those using a differentreporter gene and/or for detecting a different molecule, for instance, adifferent protease, for multiplexing. The assay may also be used todetect infectious agents, e.g., for clinical diagnostic applications, aswell as to detect bioweapons.

In one embodiment, a fusion of a mutant hydrolase and another protein isemployed for chromatin immunoprecipitation. A fusion comprising a mutanthydrolase and a DNA binding protein is expressed in a cell. After aperiod of incubation, cells are fixed, sonicated and chromatin-hybridprotein complexes are isolated with a solid support having a hydrolasesubstrate or cells are lysed by sonication, and chromatin complexes areisolated by using a hydrolase substrate attached to a solid support.Unbound complexes or proteins are removed by washing followed bycrosslinking the fusion protein to the chromatin or hydrolase substratecomprising a functional group, such as biotin, is added to the cells andincubated for a certain period of time. Cells are then fixed, sonicatedand the chromatin complexes isolated with a solid support, e.g., onelinked to streptavidin. An amplification reaction is employed tocharacterize the isolated chromatin.

In yet another embodiment, a fusion of a mutant hydrolase and a nucleicacid binding protein is employed in an in vitro nucleic acid bindingassay. The fusion is immobilized onto a solid phase which contains ahydrolase substrate. Cell lysates or purified nucleic acids areincubated the immobilized fusion protein and bound nucleic acids aredetected by gel electrophoresis or a polymerase reaction. Alternatively,the fusion is immobilized onto an electrochemically sensitive surfacecontaining a hydrolase substrate. Nucleic acid binding is determined byan electrochemical alteration. These methods could be used for highthroughput as well as multiplexed assays for two or more nucleic acidbinding proteins

In another embodiment, three vectors are employed: one vector expressesa GAL4, a protease recognition site, and VP16 fusion; a second vectorincludes a promoter linked to a GAL4 binding site linked to atranscription repressor protein gene; and a third vector which includesa promoter linked to a transcription repressor protein binding site(s)linked to the reporter gene (FIG. 60B). Binding of the GAL4 fusion tothe GAL4 binding site results in the constitutive transcriptionalactivation of RNA polymerase. When the transcription repressor proteinis being constitutively expressed, the expression of the reporter geneis inhibited. However, in presence of the protease, GAL4 and VP16 areseparated and the transcription repressor protein is not synthesized.This results in the expression of the reporter gene. In otherembodiments, a split ubiquitin (see U.S. Pat. No. 5,503,977) or adenylcyclase, guanyl cyclase and/or modulator thereof (see U.S. Pat. No.6,333,154) system may be employed. Such a system may be used formultiplexed assays using a combination of two or more differentreporters such as luciferase and GFP or luciferase and a mutanthydrolase, multiplexed assays for proteases, e.g., using combinations oftwo or more protease recognition sites, for protease, e.g., caspase,inhibitor screening assays. The assay may be used to detect infectiousagents, for instance, in clinical diagnostic applications as well as todetect bioweapons.

In a further embodiment, a cell based assay that employs reporters suchas a mutant hydrolase or luciferase with short or shortened half-livesdue to the presence of degradation/instability domains (a “proteindestabilization sequence”) is employed to detect one or more proteases(FIG. 60C). A protease, e.g., a caspase, site is introduced between thereporter protein and the protein destabilization domain(s). In theabsence of the protease, the reporter protein is rapidly degraded. Inpresence of the protease, the destabilization domain is removedresulting in a reporter protein with a longer half-life. Such a systemmay be used for multiplexed assays using a combination of two or moredifferent reporters such as luciferase and GFP or luciferase and mutanthydrolase, multiplexed assays using a combination of two or moreproteases, or for protease or caspase inhibitor screening assays.

In one embodiment, intracellular movements may be monitored using afusion of the mutant hydrolase of the invention. For example,beta-arrestin is a regulator of G-protein coupled receptors, that movesfrom the cytoplasm to the cell membrane when it is activated. A cellcontaining a fusion of a mutant hydrolase and beta-arrestin and asubstrate of the invention allows the detection of the movement ofbeta-arrestin from the cytoplasm to the cell membrane as it associateswith activated G-protein coupled receptors. The assay may be used todetect infectious agents, and so may be employed in clinical diagnosticapplications as well as to detect bioweapons.

Other formats may be used to detect proteases such as caspases. In oneembodiment, a fusion of a mutant hydrolase and another reporter protein,e.g., a luciferase, is constructed by incorporating a protease site atthe junction of the fusion. This fusion protein is immobilized onto asolid support and used for the detection of proteases in a sample. Asolid phase with the fusion protein is incubated with test samplelysate(s) and/or isolated protease(s). After a certain period ofincubation, the lysate is removed and assayed for the presence of thereporter protein. In the presence of the protease, the reporter protein,e.g., luciferase, is released from the solid support into solution. Thisassay may be used in a protein microarray or a multi-well format and inconjunction with other assays, including those using a differentreporter gene and/or for detecting a different molecule, for instance, adifferent protease, for multiplexing. The method could also be used forthe detection of infectious agents, and thus useful for clinicaldiagnostic applications, as well as for the detection of bioweapons.

In another embodiment, FRET may be employed with a fusion of the mutanthydrolase and a fluorescent protein, e.g., GFP, or a fusion with aprotein that binds fluorescent molecules, e.g., O-alkylguanine-DNAalkyltransferase (AGT) (Keppler et al., 2003). Alternatively, a fusionof a mutant hydrolase and a protein of interest and a second fusion of afluorescent protein and a molecule suspected of interacting with theprotein of interest may be employed to study the interaction of theprotein of interest with the molecule, e.g., using FRET. One cell maycontain the fusion of a mutant hydrolase and a protein of interest whileanother cell may contain the second fusion of a fluorescent protein anda molecule suspected of interacting with the protein of interest. Apopulation with those two cells may be contacted with a substrate and anagent, e.g., a drug, after which the cells are monitored to detect theeffect of agent administration on the two populations. In oneembodiment, a fusion of a mutant hydrolase and a protein of interestwhich protein of interest interacts with a second protein, and a secondfusion comprising the second protein and a mutant hydrolase may beemployed to study the interaction of the protein of interest and thesecond protein or to detect a molecule which interacts with one or bothproteins and alters their interaction, e.g., PKA regulatory subunit, PKAcatalytic subunit and cAMP. In this embodiment, two substrates with atleast one different functional group may be employed.

In yet another embodiment, the mutant hydrolase is fused to afluorescent protein. The fusion protein can thus be detected in cells bydetecting the fluorescent protein or by contacting the cells with asubstrate of the invention and detecting the functional group in thesubstrate. The detection of the fluorescent protein may be conductedbefore the detection of the functional group. Alternatively, thedetection of the functional group may be conducted before the detectionof the fluorescent protein. Moreover, those cells can be contacted withadditional substrates, e.g., those having a different functional group,and the different functional group in the cell detected, whichfunctional group is covalently linked to mutant hydrolase not previouslybound by the first substrate.

In yet another embodiment, a fusion of a mutant hydrolase and atranscription factor may be employed to monitor activation oftranscription activation pathways. For example, a fusion of a mutanthydrolase to a transcription factor present in the cytoplasm in aninactive form but which is translocated to the nucleus upon activation(e.g., NF kappa Beta) can monitor transcription activation pathways.

In another embodiment, biotin is employed as a functional group in asubstrate and the fusion includes a mutant hydrolase fused to a proteinof interest suspected of interacting with another molecule, e.g., aprotein, in a cell. The use of such reagents permits the capture of theother molecule which interacts in the cell with the protein fused to themutant hydrolase, thereby identifying and/or capturing (isolating) theinteracting molecule(s).

In one embodiment, the mutant hydrolase is fused to a protein that issecreted. Using that fusion and a substrate of the invention, thesecreted protein may be detected and/or monitored. Similarly, when themutant hydrolase is fused to a membrane protein that is transportedbetween different vesicular compartments, in the presence of thesubstrate, protein processing within these compartments can be detected.In yet another embodiment, when the mutant hydrolase is fused to an ionchannel or transport protein, or a protein that is closely associatedwith the channel or transport protein, the movement of ions across cellor organelle membranes can be monitored in the presence of a substrateof the invention which contains an ion sensitive fluorophore. Likewise,when the mutant hydrolase is fused to proteins associated with vesicalsor cytoskeleton, in the presence of the substrate, transport of proteinsor vesicals along cytoskeletal structures can be readily detected.

In another embodiment, the functional group is a drug or toxin. Bycombining a substrate with such a functional group with a fusion of amutant hydrolase and a targeting molecule such as an antibody, e.g., onewhich binds to an antigen associated with specific tumor cells, a drugor toxin can be targeted within a cell or within an animal.Alternatively, the functional group may be a fluorophore which, whenpresent in a substrate and combined with a fusion of a mutant hydrolaseand a targeting molecule such as a single chain antibody, the targetingmolecule is labeled, e.g., a labeled antibody for in vitro applicationssuch as an ELISA.

In yet another embodiment, when fused to a protein expressed on the cellsurface, a mutant hydrolase on the cell surface, when combined with asubstrate of the invention, e.g., one which contains a fluorophore, maybe employed to monitor cell migration (e.g., cancer cell migration) invivo or in vitro. In one embodiment, the substrate of the invention isone that has low or no permeability to the cell membrane. Alternatively,such a system can be used to monitor the effect of different agents,e.g., drugs, on different pools of cells. In yet another embodiment, themutant hydrolase is fused to a HERG channel. Cells expressing such afusion, in the presence of a substrate of the invention which includes aK+-sensitive fluorophore, may be employed to monitor the activity of theHERG channel, e.g., to monitor drug-toxicity. In a further embodiment,such a fusion may be expressed on the surface of blood cells, such asexogenous cells introduced to an animal or endogenous cells in atransgenic animal the genome of which is modified to express such afusion protein.

In another embodiment, the substrate of the invention includes afunctional group useful to monitor for hydrophobic regions, e.g., NileRed, in a cell or organism.

Thus, the mutant hydrolases and substrates of the invention are usefulin a wide variety of assays, e.g., phage display, panning, ELISA, massspectrometry, Western blot, fluorometric microvolume assay technology(FMAT), whole animal imaging, X-ray imaging, and cell and subcellularstaining, or as a biosensor. For example, cells expressing or containinga mutant hydrolase or a fusion protein which includes a mutanthydrolase, are introduced, e.g., implanted or injected into an animalsuch as a human or non-human animal including a non-human mammal ornon-human primate. The cells may be transiently transfected or stablyexpress the mutant hydrolase or fusion thereof, or be otherwisecontacted with the mutant hydrolase or fusion thereof so that it isassociated with the cell. Different cell types can be used, includingbut not limited to cell lines, primary cultures, or stem cells (e.g.,embryonic or adult stem cells). In one embodiment, the mutant hydrolaseexpressing or containing cells are contacted with a hydrolase substrateof the invention before introduction to an animal. In anotherembodiment, a hydrolase substrate of the invention is introduced to theanimal before or after the mutant hydrolase expressing or containingcells are introduced to the animal. The presence, location or amount ofthe functional group of the hydrolase substrate in the whole animal orin tissue preparations (including but not limited to tissue biopsy orslices, cells isolated from a physiological sample, or in homogenizedtissue), or physiological fluid samples such as blood samples and thelike, is detected or determined. The mutant hydrolase, a fusioncomprising the mutant hydrolase, and/or one or more substrates of theinvention can be used alone or in combination with other optical ornuclear reporting systems (e.g., fluorescent proteins, luciferases,radionuclides, etc.), for instance, to image biological processes, toimage transcriptional regulation of endogeneous genes, and to imagetrafficking of cells (bone marrow-derived cells, blood cells, and thelike). Optical imaging systems include those for microscopic resolution,e.g., epi, confocal and two photon, mesoscopic resolution, e.g., opticalprojection tomography, optical coherence tomography or laser speckleimaging, and macroscopic resolation with intrinsic contrast or molecularcontrast, e.g., hyperspectral imaging, endoscopy, polarization imaging,fluorescence reflectance imaging, diffuse optical tomography,fluorescence resonance imaging, fluorescence molecular imaging orluminescence imaging.

The mutant hydrolase, a fusion comprising the mutant hydrolase, and/orone or more substrates of the invention can be also used in combinationwith different optically dense/contrast reagents, which may be employedas a separate agent or chemically attached to a hydrolase substrate. Inone embodiment, a hydrolase substrate containing a contrast agent isintroduced to an animal which contains cells expressing the mutanthydrolase or a fusion thereof (e.g., a transgenic animal harboring thegene coding for mutant hydrolase or fusion thereof). In anotherembodiment, a hydrolase substrate containing a contrast agent isintroduced to cells expressing the mutant hydrolase or a fusion thereofand those cells are introduced to an animal. The contrast agent can thenbe detected using X-ray, MRI, or other techniques.

In one embodiment, a fusion of a mutant hydrolase and another proteinand a hydrolase substrate bound to an electrochemically sensitivesurface are employed to detect a molecule such as a physiologicalmolecule, i.e., they are employed as a biosensor. For instance, thesurface contains immobilized hydrolase substrate, and the presence of amolecule of interest in a test solution is determined by anelectrochemical alteration. For example, a fusion comprising a mutanthydrolase and glucose oxidase is immobilized onto a platinum electrode,gold surface, gold nanoparticles or carbon nanotubes, having a hydrolasesubstrate. A test sample is added and the presence or quantity ofglucose in the test sample determined. Likewise, cholesterol in a testsample may be determined using a mutant hydrolase-cholesterol oxidasefusion immobilized onto an electrochemically sensitive surface.

In another embodiment, the mutant hydrolase may be used as a biosensorfor the detection of protease, protease inhibitor, kinase or kinaseinhibitor and the like. For example, a protease site is fused to amutant hydrolase protein and the resulting fusion immobilized onto anelectrochemically sensitive surface such as electrode, a gold surface,gold nanoparticle, or carbon nanotube, having a hydrolase substrate. Inpresence of molecules such as a protease or kinase, there is a shift inthe molecular weight, which may be detected by an electrochemicalalteration. Inhibitors of those changes include inhibitors of theprotein fused to the mutant hydrolase, e.g., protease inhibitors, whichmay also be detected using this method.

In another embodiment, a mutant hydrolase conjugated to a substrateother than hydrolase substrate, e.g., at the C-terminal end of themutant hydrolase and/or a fusion of a mutant hydrolase and a proteinconjugated to a substrate other than the hydrolase substrate. Thebiosensor surface contains immobilized hydrolase substrate. The presenceof a biomolecule in the test solution is determined by anelectrochemical alteration. The method may be used to capture, bind orotherwise provide a means for assaying certain molecules and could beused for the detection of pesticides, industrial toxic compounds,clinical diagnostics, infectious agents and bioweapons. In oneembodiment, this method could be used for the detection of moleculesincluding, but not limited to, a protease, protease inhibitor, kinase,kinase inhibitor, as well as the detection of the post-translationalmodification of proteins.

The invention will be further described by the following non-limitingexamples.

Example I General Methodologies

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe field of molecular biology and cellular signaling and modeling.Generally, the nomenclature used herein and the laboratory procedures inspectroscopy, drug discovery, cell culture, molecular genetics, plasticmanufacture, polymer chemistry, diagnostics, amino acid and nucleic acidchemistry, and alkane chemistry described below are those well known andcommonly employed in the art. Standard techniques are typically used forpreparation of plastics, signal detection, recombinant nucleic acidmethods, polynucleotide synthesis, and microbial culture andtransformation (e.g., electroporation, lipofection).

The techniques and procedures are generally performed according toconventional methods in the art and various general references (seegenerally, Sambrook et. al. Molecular Cloning: A laboratory manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., and Lakowicz, J. R. Principles of Fluorescence Spectroscopy, NewYork: Plenum Press (1983) for fluorescent techniques, which areincorporated herein by reference) and which are provided throughout thisdocument. Standard techniques are used for chemical synthesis, chemicalanalysis, and biological assays.

Materials

All oligonucleotides were synthesized, purified and sequenced by PromegaCorporation (Madison, Wis.) or the University of Iowa DNA Facility (IowaCity, Iowa). Restriction enzymes and DNA modifying enzymes were obtainedfrom Promega Corporation (Madison, Wis.), New England Biolabs, Inc.(Beverly, Mass.) or Stratagene Cloning Systems (La Jolla, Calif.), andwere used according to the manufacturer's protocols. Competent E. coliJM109 were provided by Promega Corporation or purchased from StratageneCloning Systems. Small-scale plasmid DNA isolations were done using theQiagen Plasmid Mini Kit (Qiagen Inc., Chatsworth, Calif.). DNA ligationswere performed with pre-tested reagent kits purchased from StratageneCloning Systems. DNA fragments were purified with QIAquick GelExtraction Kits or QIAquick PCR purification Kits purchased from QiagenInc.

The vectors used for generating DhaA mutants and their fusions were asfollows: pET21 (Invitrogen, Carlsbad, Calif.), pRL-null (Promega,Madison, Wis.), pGEX-5x-3 (Amersham Biosciences; Piscataway, N.J.), andEGFP and DsRED2 (both from CLONTECH, Palo Alto, Calif.).

SDS-polyacrylamide gels and associated buffers and stains, as well aselectroblot transfer buffers, were obtained from BioWhittaker MolecularApplications (Rockland, Me.). Protein molecular weight standards werepurchased from Invitrogen.

Sigma-Aldrich was the source of Anti Flag® monoclonal antibodyantibodies (anti FLAG® M2 monoclonal antibody (mouse) (F3165)), AntiFLAG® M2 HRP Conjugate and Anti FLAG® M2 FITC conjugate (A8592 andF4049, respectively). Chemicon (Temecula, Calif.) was the source ofmonoclonal anti-Renilla luciferase antibody (MAB4410). Promega Corp. wasthe source of HRP-conjugated goat anti-mouse IgG and HRP-conjugatedstreptavidin (W4021 and G714, respectively).

1-Cl-butane, 1-Cl-hexane, 1-Cl-octane, 1-Cl-decane, 1-Cl-butanol,1-Cl-hexanol, 1-Cl-octanol, and 1-Cl-decanol were obtained from Aldrichor from Fluka (USA). All salts, monobasic potassium phosphate, dibasicpotassium phosphate, imidazole, HEPES, sodium EDTA, ammonium sulfate,and Tris free base were from Fisher (Biotech Grade).

Glutathione Sepharose 4 FF, glutathione, MonoQ and Sephadex G-25prepackaged columns were from Amersham Biosciences.

Luria-Broth (“LB”) was provided by Promega Corporation.

Methods

PCR Reactions.

DNA amplification was performed using standard polymerase chain reactionbuffers supplied by Promega Corp. Typically, 50 μl reactions included 1×concentration of the manufacturer's supplied buffer, 1.5 mM MgCl₂, 125μM dATP, 125 μM dCTP, 125 μM dGTP, 125 μM dTTP, 0.10-1.0 μM forward andreverse primers, 5 U AmpliTaq® DNA Polymerase and <1 ng target DNA.Unless otherwise indicated, the thermal profile for amplification of DNAwas 35 cycles of 0.5 minutes at 94° C.; 1 minute at 55° C.; and 1 minuteat 72° C.

DNA Sequencing.

All clones were confirmed by DNA sequencing using the dideoxy-terminalcycle-sequencing method (Sanger et al., 1977) and a Perkin-Elmer Model310 DNA sequencer. (Foster City, Calif.).

SDS-PAGE.

Proteins were solubilized in a sample buffer (1% SDS, 10% glycerol, and1.0 mM ↑-mercaptoethanol, pH 6.8; Promega Corporation), boiled for 5minutes and resolved on SDS-PAGE (4-20% gradient gels; BioWhittakerMolecular Applications). Gels were stained with Coomassie Blue (PromegaCorp.) for Western blot analysis or were analyzed on a fluoroimager(Hitachi, Japan) at an E_(ex)/E_(em) appropriate for each fluorophoreevaluated.

Western Blot Analysis.

Electrophoretic transfer of proteins to a nitrocellulose membrane (0.2μm, Scheicher & Schuell, Germany) was carried out in 25 mM Tris base/188mM glycine (pH 8.3), 20% (v/v) methanol for 2.0 hours with a constantcurrent of 80 mA (at 4° C.) in Xcell II Blot module (Invitrogen). Themembranes were rinsed with TBST buffer (10 mM Tris-HCl, 150 mM NaCl, pH7.6, containing 0.05% Tween 20) and incubated in blocking solution (3%dry milk or 1% BSA in TBST buffer) for 30 minutes at room temperature orovernight at 4° C. Then membranes were washed with 50 ml of TBST bufferand incubated with anti-FLAG® monoclonal antibody M2 (dilution 1:5,000),anti-Renilla luciferase monoclonal antibody (dilution 1:5,000), orHRP-conjugated streptavidin (dilution 1:10,000) for 45 minutes at roomtemperature. Then the membranes were washed with TBST buffer (50 ml, 5minutes, 3 times). The membranes that had been probed with antibody werethen incubated with HRP-conjugated donkey anti-mouse IgG (30 minutes,room temperature) and then the washing procedure was repeated. Theproteins were visualized by the enhanced chemiluminescence (ECL) system(Pharmacia-Amersham) according to the manufacturer's instructions.Levels of proteins were quantified using computer-assisted densitometry.

Protein Concentration.

Protein was measured by the microtiter protocol of the Pierce BCAProtein assay (Pierce, Rockford, Ill.) using bovine serum albumin (BSA)as a standard.

Statistic Analysis.

Data were expressed as mean+/−S.E.M. values from experiments performedin quadruplicate, representative of at least 3 independent experimentswith similar results. Statistical significance was assessed by thestudent's t test and considered significant when p<0.05.

Bacterial Cells.

The initial stock of Dh5α cells containing pET-3a with Rhodococcusrodochorus (DhaA) was kindly provided by Dr. Clifford J. Unkefer (LosAlamos National Laboratory, Los Alamos, N. Mex.) (Schindler et al.,1999; Newman et al., 1999). Bacteria were cultured in LB using apremixed reagent provided by Promega Corp. Freezer stocks of E. coliBL21 (λDE3) pET3a (stored in 10% glycerol, −80° C.) were used toinoculate Luria-Bertani agar plates supplemented with ampicillin (50ng/ml) (Sambrook et al., 1989). Single colonies were selected and usedto inoculate two 10 ml cultures of Luria-Bertani medium containing 50ng/ml ampicillin. The cells were cultured for 8 hours at 37° C. withshaking (220 rpm), after which time 2 ml was used to inoculate each oftwo 50 ml of Luria-Bertani medium containing 50 ng/ml ampicillin, whichwere grown overnight at 37° C. with shaking. Ten milliliters of thisculture was used to inoculate each of two 0.5 L Luria-Bertani mediumwith ampicillin. When the A₆₀₀ of the culture reached 0.6,isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to a finalconcentration of 0.5 mM, and cultures were maintained for an additional4 hours at 30° C. with shaking. The cells were then harvested bycentrifugation and washed with 10 mM Tris-SO₄, 1 mM EDTA, pH 7.5. Thecell pellets were stored at −70° C. prior to cell lysis.

Mammalian Cells.

CHO-K1 cells (ATCC-CCL61) were cultured in a 1:1 mixture of Ham's F12nutrients and Dulbecco's modified minimal essential medium supplementedwith 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/mlstreptomycin, in an atmosphere of 95% air and 5% CO₂ at 37° C.

Rat hippocampal (E18) primary neurons were isolated as described below.Briefly, fragments of embryonic (E18) rat hippocampus in Hibernate™ Emedia (GIBCO, Invitrogen, Carlsbad, Calif.), obtained from Dr. Brewer(Southern Illinois University), were dissociated and plated onpoly-D-lysin coated (0.28 mg/cm²; Sigma) glass/plastic-ware and culturedin serum-free Neurobasal™ media with B27 supplement (NB27, GIBCO). Allmedia were changed every 2-3 days.

Transfection.

To study transient expression of different proteins, cells were platedin 35 mm culture dishes or 24 well plates. At about 80-90% confluency,the cells were exposed to a mixture of lipofectamine/DNA/antibiotic freemedia according to the manufacturer's (GIBCO) instructions. Thefollowing day, media was replaced with fresh media and cells wereallowed to grow for various periods of time.

Fluorescence.

Fluorescence in cells in 96 well plates was measured on fluorescentplate reader CytoFluorII (Beckman) at an E_(ex)/E_(em) appropriate forparticular fluorophores (e.g., E_(ex)/E_(em) forcarboxytetramethylrhodamine is 540/575 nm).

Example II A DhaA-Based Tethering System

A. Wild-Type and Mutant DhaA Proteins and Fusions Thereof

The haloalkane dehalogenase gene from Rhodococcus rhodochrous, dhaA,encodes a monomeric 33 kDa enzyme that catalyzes the irreversiblehydrolysis of a variety of haloalkanes (Kulakova et al. 1997), e.g.,cleaves carbon-halogen bonds in aliphatic and aromatic halogenatedcompounds, e.g., HaloC₃-HaloC₁₀. A substantial amount of mechanistic andstructural information is available on the haloalkane dehalogenases. TheDhaA enzyme contains 293 amino acids and is a member of a superfamily ofproteins containing an α/β hydrolase fold (FIG. 2A). The overallstructures of the haloalkane dehalogenases from Rhodococcus,Xanthobacter and Sphingomonas are similar and each contains a triad ofcatalytic residues that is involved in the cleavage of halide-carbonbonds. In the case of DhaA, these residues are Asp106, E130, and His272(Newman et al., 1999; FIG. 2B). FIGS. 1A-B show the overall catalyticmechanism for the DhaA enzyme. After substrate binding, nucleophilicattack by the carboxylate of an Asp residue on the substrate causes thecleavage of the halogen-carbon bond and the formation of an alkyl-esterintermediate (FIG. 1A). The next step in the dehalogenase reactionpathway is hydrolysis of the intermediate ester by a water moleculeactivated by the active site His residue (FIG. 1B). While the catalytichistidine residue is the base catalyst for the dealkylation of thecovalent intermediate, it is not essential for the initial nucleophilicattack of the active site Asp. Protein variants that lack the crucialcatalytic histidine residue have been shown to carry out the alkylationhalf reaction thereby producing a stable, covalent ester intermediate(Pries et al., 1995).

It is likely that substrate binds to DhaA to form an E*S complex, afterwhich nucleophilic attack by Asp106 forms an ester intermediate, His272then activates H₂O that hydrolyzes the intermediate, releasing productfrom the catalytic center. To determine whether a point mutation of thecatalytic His272 residue impairs enzymatic activity of the enzyme so asto enable covalent tethering of a functional group (FG) to this protein,mutant DhaAs were prepared.

Materials and Methods

To prepare mutant DhaA vectors, Promega's in vitro mutagenesis kit whichis based on four primer overlap-extension method was employed (Ho etal., 1989) to produce DhaA.H272 to F, A, G, or H mutations. The externalprimers were oligonucleotides

(SEQ ID NO: 1) 5′-GCTTCACTTGTCGTCATCGTCCTTGTAGTCA-3′   and(SEQ ID NO: 2) 5′-GCTTCACTTGTCGTCATCGTCCTTGTAGTCA-3′,and the internal mutagenic primers were as follows:

H272F  (SEQ ID NO: 3) 5′-CCGGGATTGTTCTACCTCCAGGAAGAC- 3′,  H272A(SEQ ID NO: 4) 5′-CCGGGATTGGCCTACCTCCAGGAAGAC-3′; H272G  (SEQ ID NO: 5)5′-CCGGGATTGCAGTACCTCCAGGAAGAC-3′;   and H272Q  (SEQ ID NO:6)5′-CCGGGATTGGGCTACCTCCAGGAAGAC-3′; (the mutated codons are underlined). The mutated dehalogenase genes weresubcloned into the pET-3a vector. For overexpression of mutantdehalogenases, the pET-3a vector was transformed into competent E. coliBL21 (DE3). The DhaA sequence in clones was confirmed by DNA sequencing.Unless otherwise noted DhaA.WT and DhaA.H272F proteins generally containGST at the N-terminus and a FLAG epitope at the C-terminus.

GST-DhaA (WT or H272F/A/G/H mutants) fusion cassettes were constructedby cloning the appropriate DhaA coding regions into SalI/NotI sites ofpGEX5x3 vector. Two primers

(SEQ ID NOs:7 and 8, 5′-ACGCGTCGACGCCGCCATGTCAGAAATCGGTACAGGC-3′ and 5′-ATAAGAATGCGGCCGCTCAAGCGCTTCAACCGGTGAGTGCGGGGAGC  CAGCGCGC-3′;respectively) were designed to add a SalI site and a Kozak consensussequence to the 5′ coding regions of DhaA, to add a NotI, EcoR47III, andAgeI restriction site and stop codons to the 3′ coding region of DhaA,and to amplify a 897 bp fragment from a DhaA (WT or mutant) template.The resulting fragments were inserted into the SalI/NotI site ofpGEX-5X-3, a vector containing a glutathione S-transferase (GST) gene, asequence encoding a Factor Xa cleavage site, and multiple cloning sites(MCS) followed by a stop codon.

A Flag coding sequence was then inserted into the AgeI/EcoR47IIIrestriction sites of the pGEX5X-3 vector. In frame with the sixnucleotide AgeI site is a sequence for an 11 amino acid peptide, thefinal octapeptide of which corresponds to the Flag peptide (KodakImaging Systems, Rochester, N.Y.). Two complementary oligonucleotides

(5′-CCGGTGACTACAAGGACGATGACGACAAGTGAAGC-3′, sense, SEQ ID NO: 9, and5′-GCTTCACTTGTCGTCATCGTCCTTGTAGTCA-3′, antisense, SEQ ID NO: 10)coding the Flag peptide (Kodak Imaging Systems, Rochester, N.Y.) wereannealed. The annealed DNA had an AgeI site at the 5′ end and anEcoR47III at the 3′ end. The annealed DNA was digested with AgeI andEcoR47III and then subcloned into the GST-DhaA.WT or GST-DhaA.H272Fmutant constructs at the AgeI and EcoR47III sites. All gene fusionconstructs were confirmed by DNA sequencing. Unless otherwise notedDhaA.WT and DhaA.H272F proteins generally contain GST at the N-terminusand a FLAG epitope at the C-terminus.

To generate DhaA fusion proteins, enzyme expression was induced by theaddition of isopropyl-b-D-thiogalactopyranoside (at a finalconcentration of 0.5 mM) when the culture reached an optical density of0.6 at 600 nm. The cells were harvested in Buffer A (10 mM Tris-SO₄, 1mM EDTA, 1 mM β-mercaptoethanol, and 10% glycerol, pH 7.5), anddisrupted by sonication using a Vibra Cell™ sonicator (Sonics &Materials, Danbury, Conn., USA). Cell debris was removed bycentrifugation at 19,800×g for 1 hour. The crude extract was furtherpurified on a GSS-Sepharose 4 fast flow column (Amersham Biosciences;Piscataway, N.J.) according to the manufacturer's instructions. Theelution fractions containing GST-DhaA fusion protein were pooled,dialyzed against a 10 mM Tris-SO₄ buffer (containing 20 mM Na₂SO₄ and 1mM EDTA-Na₂) overnight at 4° C., and stored at −20° C. until use. Togenerate DhaA (WT or mutant), GST was cleaved from the fusion proteinswith Factor Xa, and the products purified on GSS-Sepharose 4 (AmershamBiosciences; Piscataway, N.J.) according to the manufacturer'sinstructions. Homogeneity of the proteins was verified by SDS-PAGE. Insome experiments, the cell free extract was fractionated using 45-70%saturated ammonium sulfate as described by Newman et al. (1999).

Results

FIG. 3 shows robust, IPTG inducible production of DhaA.WT (lane 1) andDhaA.H272F (lane 2) fusion proteins. Moreover, the proteins were solubleand could be efficiently purified on Glutathione-Sepharose 4FF (lanes5-10, odd numbered lanes correspond to DhaA.WT and even numbered lanescorrespond to DhaA.H272F). Treatment of the fusion proteins with FactorXa led to the formation of two proteins GST and DhaA (WT or H272Fmutant, lanes 11 and 12, respectively), and GST was efficiently removedon Glutathione-Sepharose 4FF (WT or mutant, lanes 13 and 14,respectively). In addition, all proteins had the predicted molecularweight.

B. Mutation of H272 Impairs Ability of DhaA to Hydrolyze Cl-Alkanes.

Inability of an enzyme to release product of the enzymatic reaction intosurrounding media is essential for the tethering system. This inabilitycan be detected by significant reduction of the hydrolytic activity ofthe enzyme.

To study the effect of a point mutation on the activity of DhaA (WT ormutant) hydrolysis of Cl-alkanes, a pH-indicator dye system as describedby Holloway et al. (1998) was employed.

Materials and Methods

The reaction buffer for a pH-indicator dye system consisted of 1 mMHEPES-SO₄ (pH 8.2), 20 mM Na₂SO₄, and 1 mM EDTA. Phenol red was added toa final concentration 25 μg/ml. The halogenated compounds were added toapparent concentrations that could insure that the dissolved fraction ofthe substrate was sufficient for the maximum velocity of thedehalogenation reaction. The substrate-buffer solution was vigorouslymixed for 30 seconds by vortexing, capped to prevent significantevaporation of the substrate and used within 1-2 hours. Prior to eachkinetic determination, the phenol red was titrated with a standardizedsolution of HCl to provide an apparent extinction coefficient. Thesteady-state kinetic constants for DhaA were determined at 558 nm atroom temperature on a Beckman Du640 spectrophotometer (Beckman Coulter,Fullerton, Calif.). Kinetic constants were calculated from initial ratesusing the computer program SigmaPlot. One unit of enzyme activity isdefined as the amount required to dehalogenate 1.0 mM ofsubstrate/minute under the specific conditions.

Results

As shown in FIG. 4, using 0.1 mg/ml of enzyme and 10 mM substrate at pH7.0-8.2, no catalytic activity was found with any of four mutants. Underthese conditions, the wild-type enzyme had an activity with 1-Cl-butaneof 5 units/mg of protein. Thus, the activity of the mutants was reducedby at least 700-fold.

Aliquots of the supernatant obtained from E. coli expressing DhaA (WT orone of the mutants) were treated with increasing concentrations of(NH₄)₂SO₄. The proteins were exposed to each (NH₄)₂SO₄ concentration for2 hours (4° C.), pelleted by centrifugation, dialyzed overnight againstbuffer A, and resolved on SDS-PAGE.

As shown in FIG. 5, a major fraction of DhaA.WT and the DhaA.H272Fmutant was precipitated by 45-70% of (NH₄)₂SO₄. No precipitation ofthese proteins was observed at low (NH₄)₂SO₄ concentrations. Incontrast, the DhaA.H272Q, DhaA.H272G and DhaA.H272A mutants could beprecipitated by 10% (NH₄)₂SO₄. This is a strong indication of thesignificant change of the physico-chemical characteristics of theDhaA.H272Q, DhaA.H272G and DhaA.H272A mutants. At the same time, theDhaA.H272F mutation had no significant effect on these parameters. Thesedata are in good agreement with results of computer modeling of theeffect of mutations on the 3-D structure of DhaA, indicating that amongall tested mutants, only the DhaA.H272F mutation had no significanteffect on the predicted 3-dimensional model (see FIG. 2). Based on theseresults, DhaA.H272F was chosen for further experiments.

To form a covalent adduct, the chlorine atom of Cl-alkane is likelypositioned in close proximity to the catalytic amino acids of DhaA (WTor mutant) (FIGS. 2A-B). The crystal structure of DhaA (Newman et al.,1999) indicates that these amino acids are located deep inside of thecatalytic pocket of DhaA (approximately 10 Å long and about 20 Å² incross section). To permit entry of the reactive group in a substrate forDhaA which includes a functional group into the catalytic pocket ofDhaA, a linker was designed to connect the Cl-containing substrate witha functional group so that the functional group is located outside ofthe catalytic pocket, i.e., so as not to disturb/destroy the 3-Dstructure of DhaA.

To determine if DhaA is capable of hydrolyzing Cl-alkanes with a longhydrophobic carbon chain, DhaA.WT was contacted with various Cl-alkanealcohols. As shown in FIG. 6, DhaA.WT can hydrolyze 1-Cl-alkane alcoholswith 4-10 carbon atoms. Moreover, the initial rate of hydrolysis (IRH)of Cl-alkanes had an inverse relationship to the length of a carbonchain, although poor solubility of long-chain Cl-alkanes in aqueousbuffers may affect the efficiency of the enzyme-substrate interaction.Indeed, as shown in FIG. 6, the IRH of 1-Cl-alkane-10-decanol is muchhigher than the IRH of 1-Cl-decane. More importantly, these dataindicate that DhaA can hydrolyze Cl-alkanes containing relatively polargroups (e.g., HO-group).

Carboxyfluorescein-modified Cl-alkanes with linkers of different lengthand/or hydrophobicity were prepared (FIG. 7). DhaA.WT efficientlyhydrolyzed Cl-alkanes with a relatively bulky functional group(carboxyfluorescein) if the linker was 12 or more atoms long. Noactivity of DhaA.H272F/A/G/Q mutants was detected with any of the testedCl-alkanes (data not shown). In addition, modification of the (CH₂)₆region adjacent to the Cl-atom led to a significant reduction of the IRHof the 14-atom linker by DhaA.WT. Nevertheless, if the length andstructure of the linker is compatible with the catalytic site of ahydrolase, the presence of a linker in a substrate of the invention hassubstantially no effect on the reaction.

Some of the samples were analyzed on an automated HPLC (Hewlett-PackardModel 1050) system. A DAD detector was set to record UV-visible spectraover the 200-600 nm range. Fluorescence was detected at an E_(ex)/E_(em)equal 480/520 nm and 540/575 nm for carboxyfluorescein- andcarboxytetramethylrhodamine-modified substrates, respectively. Ethanolextracts of Cl-alkanes or products of Cl-alkane hydrolysis were analyzedusing analytical reverse phase C₁₈ column (Adsorbosphere HS, 5μ, 150×4.6mm; Hewlett-Packard, Clifton, N.J.) with a linear gradient of 10 mMammonium acetate (pH 7.0):ACN (acetonitrile) from 25:75 to 1:99 (v/v)applied over 30 minutes at 1.0 ml/minute. Quantitation of the separatedcompounds was based on the integrated surface of the collected peaks.

FIG. 8A shows the complete separation of the substrate and the productof the reaction. FIG. 8B indicates that DhaA.WT very efficientlyhydrolyzed carboxyfluorescein-C₁₀H₂₁NO₂—Cl. Similar results wereobtained when carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl or5-carboxy-X-rhodamine-C₁₀H₂₁NO₂—Cl were used as substrates (data notshown). Taken together these data confirm the results of thepH-indicator dye-based assay showing complete inactivation of DhaAcatalytic activity by the DhaA.H272F mutation.

C. Covalent Tethering of Functional Groups to DhaA Mutants In Vitro

Materials and Methods

MALDI analysis of proteins was performed at the University of WisconsinBiotechnology Center using a matrix assisted laser desorption/ionizationtime-of-life (MALDI-TOF) mass spectrometer Bruker Biflex III (Bruker,USA.). To prepare samples, 100 μg of purified DhaA (WT or H272F mutant)or GST-DhaA (WT or H272F mutant) fusion protein (purified to about 90%homogeneity) in 200 μl of buffer (1 mM HEPES-SO₄ (pH 7.4), 20 mM Na₂SO₄,and 1 mM EDTA) were incubated with or without substrate(carboxyfluorescein-C₁₀H₂₁NO₂—Cl, at 1.0 mM, final concentration) for 15minutes at room temperature. Then the reaction mixtures were dialyzedagainst 20 mM CH₃COONH₄ (pH 7.0) overnight at 4° C. and M/Z values ofthe proteins and protein-substrate complexes determined.

Oligonucleotides employed to prepare DhaA.D106 mutants include forDhaA.D106C:

(SEQ ID NO: 13) 5′-CTTGGGTTTGGAAGAGGTCGTCCTGGTCATCCACTGCTGGGGC-3′ and(SEQ ID NO: 14) 5′-TGAGCCCCAGCAGTGGATGACCAGGACGACCTCTTCCAAACC-3′;for DhaA.D106Q:

(SEQ ID NO: 34) 5′-CTTGGGTTTGGAAGAGGTCGTCCTGGTCATCCACCAGTGGGGC-3′ and(SEQ ID NO: 35) 5′-TGAGCCCCACTGGTGGATGACCAGGACGACCTCTTCCAAACC-3′;for DhaA.D106E:

(SEQ ID NO: 52) 5′-CTTGGGTTTGGAAGAGGTCGTCCTGGTCATCCACGAATGGGGC-3′ and(SEQ ID NO: 53) 5′-TGAGCCCCATTCGTGGATGACCAGGACGACCTCTTCCAAACC-3′; andfor DhaA.D106Y:

(SEQ ID NO: 54) 5′-CTTGGGTTTGGAAGAGGTCGTCCTGGTCATCCACTACTGGGGC-3′ and(SEQ ID NO: 55) 5′-TGAGCCCCAGTAGTGGATGACCAGGACGACCTCTTCCAAACC-3′.The annealed oligonucleotides contained a StyI site at the 5′ end andthe BlpI site at the 3′ end. The annealed oligonucleotides were digestedwith StyI and BlpI and subcloned into DhaA.WT or DhaA.H272F at StyI andBlpI sites. All mutants were confirmed by DNA sequencing.Results

To confirm that DhaA.H272 mutants were capable of binding Cl-alkaneswith functional groups, these mutants or their GST-fusions, as well asthe corresponding wild-type proteins or fusions, were contacted withcarboxyfluorescein-C₁₀H₂₁NO₂—Cl,carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl,5-carboxy-X-rhodamine-C₁₀H₂₁NO₂—Cl, or biotin-C₁₀H₂₁NO₂—Cl for 15minutes at room temperature. Then the proteins were resolved onSDS-PAGE. The gels containing proteins were incubated withcarboxyfluorescein-C₁₀H₂₁NO₂—Cl,carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl, or5-carboxy-X-rhodamine-C₁₀H₂₁NO₂—Cl and were analyzed by fluoroimager(Hitachi, Japan) at an E_(ex)/E_(em) appropriate for each fluorophore.Gels containing proteins incubated with biotin-C₁₀H₂₁NO₂—Cl weretransferred to a nitrocellulose membrane and probed with HRP conjugatedstreptavidin.

As shown in FIG. 9, carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (lanes 1and 2 in FIG. 9A), carboxyfluorescein-C₁₀H₂₁NO₂—Cl (lanes 3 and 4 inFIG. 9A), and 5-carboxy-X-rhodamine-C₁₀H₂₁NO₂—Cl (lanes 5 and 6 in FIG.9A) bound to DhaA.H272F (lanes 2, 4 and 6 in FIG. 9A) but not to DhaA.WT(lanes 1, 3 and 5 in FIG. 9A). Biotin-C₁₀H₂₁NO₂—Cl bound to DhaA.H272F(lanes 9-14 in FIG. 9B) but not to DhaA.WT (lanes 1-8 in FIG. 9B).Moreover, the binding of biotin-C₁₀H₂₁NO₂—Cl to DhaA.H272F (lanes 9-14in FIG. 9B) was dose dependent and could be detected at 0.2 μM. Further,the bond between substrates and DhaA.H272F was very strong, sinceboiling with SDS did not break the bond.

All tested DhaA.H272 mutants, i.e. H272F/G/A/Q, bound tocarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (FIG. 10). Further, theDhaA.H272 mutants bind the substrates in a highly specific manner, sincepretreatment of the mutants with one of the substrates(biotin-C₁₀H₂₁NO₂—Cl) completely blocked the binding of anothersubstrate (carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl) (FIG. 10).

To determine the nature of the bond between Cl-alkanes and theDhaA.H272F mutant (or the GST-DhaA.H272F mutant fusion protein), theseproteins were incubated with and withoutcarboxyfluorescein-C₁₀H₂₁NO₂—Cl, and analyzed by MALDI. As shown in FIG.11, the bond between mutant DhaA.H272F andcarboxyfluorescein-C₁₀H₂₁NO₂—Cl is strong. Moreover, the analysis of theE*S complex indicated the covalent nature of the bond between thesubstrate (e.g., carboxyfluorescein-C₁₀H₂₁NO₂—Cl) and DhaA.H272F. TheMALDI-TOF analysis also confirms that the substrate/protein adduct isformed in a 1:1 relationship.

DhaA mutants at another residue in the catalytic triad, residue 106,were prepared. The residue at position 106 in wild-type DhaA is D, oneof the known nucleophilic amino acid residues. D at residue 106 in DhaAwas substituted with nucleophilic amino acid residues other than D,e.g., C, Y and E, which may form a bond with a substrate which is morestable than the bond formed between wild-type DhaA and the substrate. Inparticular, cysteine is a known nucleophile in cysteine-based enzymes,and those enzymes are not known to activate water.

A control mutant, DhaA.D106Q, single mutants DhaA.D106C, DhaA.D106Y, andDhaA.D106E, as well as double mutants DhaA.D106C:H272F,DhaA.D106E:H272F, DhaA.D106Q:H272F, and DhaA.D106Y:H272F were analyzedfor binding to carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (FIG. 12). Asshown in FIG. 12, carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl bound toDhaA.D106C, DhaA.D106C:H272F, DhaA.D106E, and DhaA.H272F. Thus, the bondformed between carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl and cysteine orglutamate at residue 106 in a mutant DhaA is stable relative to the bondformed between carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl and DhaA.WT.Other substitutions at position 106 alone or in combination withsubstitutions at other residues in DhaA may yield similar results.Further, certain substitutions at position 106 alone or in combinationwith substitutions at other residues in DhaA may result in a mutant DhaAthat forms a bond with only certain substrates.

Example III Tethering of Luciferase to a Solid Support via a Mutant DhaAand a Substrate of the Invention

Materials and Methods

phRLuc-connector-DhaA.WT-Flag and phRLuc-connector-DhaA.H272F-Flagfusion cassettes were constructed by cloning the phRLuc coding regioninto the NheI/SalI sites of the pCIneo vector which contains a myristicacid attachment peptide coding sequence (MAS). Two primers(5′-GCTTCACTTGTCGTCATCGTCCTTGTAGTCA-3′; SEQ ID NO:11) and (5′-GCTTCACTTGTCGTCATCGTCCTTGTAGTCA-3′; SEQ ID NO:12) were designed to addNheI and SalI sites to the 5′ and 3′ coding regions, respectively, ofphRLuc and to amplify a 900 bp fragment from a phRLuc template (pGL3vector, Promega). Then, a myristic acid attachment peptide codingsequence was excised with NheI and SalI restriction enzymes and theamplified fragment containing phRLuc was inserted into the NheI/SalIrestriction sites of pCIneo.DhaA.(WT or H272F)-Flag vector. The sequenceof each construct was confirmed by DNA sequencing. Promega's TNT®T7Quick system was then used to generate fusion proteins in vitro.

Results

To demonstrate tethering of proteins to a solid support viaDhaA.H272F-Cl-alkane bridge, vectors encoding a fusion protein ofRenilla luciferase (hRLuc, N-terminus of the fusion), a proteinconnector (17 amino acids, see Table I), and DhaA (WT or H272F mutant)were prepared. The Flag epitope was then fused to the C-terminus ofDhaA.

TABLE I Peptide Fusion Sequence Connector GST-atcgaaggtcgtgggatccccaggaa iegrgiprnsr DhaA ttcccgggtcgacgccgcc vdaa(SEQ ID NO: 26) (SEQ ID NO: 27) GFP- tccggatcaagcttgggcgacgaggtsgsslgdevdg DhaA ggacggcgggccctctagagccacc gpsrat (SEQ ID NO: 28)(SEQ ID NO: 29) DhaA- accggttccggatcaagcttgcggta tgsgsslryrg Rlucccgcgggccctctagagcc psra (SEQ ID NO: 30) (SEQ ID NO: 31) Rluc-tccggatcaagcttgcggtaccgcgg sgsslryrgps DhaA gccctctagagccgtcgacgccgccravdaa (SEQ ID NO: 32) (SEQ ID NO: 33) DhaA- Accggt Tg Flag

SDS-PAGE followed by Western blot analysis showed that the proteins hadtheir predicted molecular weights and were recognized by anti-R.Luc andanti-Flag® M2 antibodies. In addition, all fusion proteins had Renillaluciferase activity (as determined by Promega's Renilla Luciferase AssaySystem in PBS pH 7.4 buffer).

Tethering of proteins to a solid support via a DhaA.H272F-Cl-alkanebridge was shown by using biotin-C₁₀H₂₁N₁O₂—Cl as a substrate andstreptavidin (SA)-coated 96 well plates (Pierce, USA) as solid support.Translated proteins were contacted with biotin-C₁₀H₂₁N₁O₂—Cl substrateat 25 μM (final concentration), for 60 minutes at room temperature.Unbound biotin-C₁₀H₂₁N₁O₂—Cl was removed by gel-filtration on SephadexG-25 prepackaged columns (Amersham Biosciences). Collected fractions ofR.Luc-connector-DhaA fusions were placed in SA-coated 96-well plate for1 hour at room temperature, unbound proteins were washed out andluciferase activity was measured.

FIG. 13A shows Renilla luciferase activity captured on the plate.Analysis of these data indicated that only the fusion containing themutant DhaA was captured. The efficiency of capturing was very high(more than 50% of Renilla luciferase activity added to the plate wascaptured). In contrast, the efficiency of capturing of fusionscontaining DhaA.WT as well as Renilla luciferase was negligibly small(<0.1%). Pretreatment of R.Luc-connector-DhaA.H272F with anon-biotinylated substrate (carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl)decreased the efficiency of capturing by about 80%. Further, there wasno effect of pretreatment with a nonbiotinylated substrate on thecapturing of the R.Luc-connector-DhaA.WT or Renilla luciferase.

Taken together, these data demonstrate that active enzymes (e.g.,Renilla luciferase) can be tethered to a solid support that forms partof a substrate of the invention (Cl-alkane-DhaA.H272F-bridge), andretain enzymatic activity.

Example IV Mutant DhaA and Substrate System In Vivo

A. Covalent Tethering of Functional Groups to DhaA Mutants In Vivo: inProkaryotes and Eukaryotes

Materials and Methods

To study the binding of a substrate of the invention to a mutanthydrolase expressed in prokaryotes, E. coli cells BL21 (λDE3) pLys65were transformed with pGEX-5X-3.DhaA.WT-Flag orpGEX-5X-3.DhaA.H272F-Flag, grown in liquid culture, and induced withIPTG. Either carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl orbiotin-C₁₀H₂₁NO₂—Cl was added to the induced cells (final concentration,25 μM). After 1 hour, cells were harvested, washed with cold PBS (pH7.3), disrupted by sonication, and fractionated by centrifugation at19,800×g for 1 hour. Soluble fractions were subjected to SDS-PAGE. Gelswith proteins isolated from cells treated withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl were analyzed on afluoroimager, while proteins from cells treated withbiotin-C₁₀H₂₁N₁O₂—Cl were transferred to a nitrocellulose membrane andprobed with HRP-conjugated streptavidin.

To study the binding of carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl inmammalian cells, DhaA.WT-Flag and DhaA.H272F-Flag coding regions wereexcised from pGEX-5X-3.DhaA.WT-Flag or pGEX-5X-3.DhaA.H272F-Flag,respectively, gel purified, and inserted into SalI/NotI restrictionsites of pCIneo.CMV vector (Promega). The constructs were confirmed byDNA sequencing.

CHO-K1 cells were plated in 24 well plates (Labsystems) and transfectedwith a pCIneo-CMV.DhaA.WT-Flag or pCIneo-CMV.DhaA.H272F-Flag vector.Twenty-four hours later, media was replaced with fresh media containing25 μM carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl and the cells were placedinto a CO₂ incubator for 60 minutes. Following this incubation, mediawas removed, cells were quickly washed with PBS (pH 7.4; fourconsecutive washes: 1.0 ml/cm²; 5 seconds each) and the cells weresolubilized in a sample buffer (1% SDS, 10% glycerol, and the like; 250μl/well). Proteins (10 μl/lane) were resolved on SDS-PAGE (4-20%gradient gels) and the binding of thecarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl was detected by a fluoroimager(Hitachi, Japan) at E_(ex)/E_(em) equal 540/575 nm.

Results

FIGS. 14A and B show the binding of biotin-C₁₀H₂₁NO₂—Cl (A) andcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (B) to E. coli proteins invivo. The low molecular band on FIG. 14A is an E. coli proteinrecognizable by HRP-SA, while the fluorescence detected in the bottompart of FIG. 14B was fluorescence of freecarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl. FIG. 15 shows the binding ofcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl to eukaryotic cell proteins invivo.

Analysis of FIG. 14 and FIG. 15 showed that the DhaA.H272F-Flag mutantbut not DhaA.WT-Flag binds carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl orbiotin-C₁₀H₂₁N₁O₂—Cl in vivo. Moreover, the bond between DhaA.H272F-Flagand the substrate was very strong (probably covalent), since boilingwith SDS followed by SDS-PAGE did not disrupt the bond between themutant enzyme and the substrate.

B. Permeability of Cell Membrane to Substrates of the Invention

Materials and Methods

CHO-K1 Cells (ATCC-CCL61) were cultured in a 1:1 mixture of Ham's F12nutrients and Dulbecco's modified minimal essential medium supplementedwith 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/mlstreptomycin, in an atmosphere of 95% air and 5% CO₂ at 37° C.

To study uptake of different substrates, cells were plated in LT-IIchambers (Nunc) or 96 well plates (Labsystems) at a density of 30,000cells/cm². The following day, media was replaced with media containingdifferent concentrations of the substrates and cells were placed back ina CO₂ incubator for 2, 5 or 15 minutes. At the end of the incubation,media containing substrate was removed and cells were quickly washedwith PBS (pH 7.4; four consecutive washes: 1.0 ml/cm²; 5 seconds each).Fresh media was then added to cells, and the cells were returned to theCO₂ incubator at 37° C. The level of fluorescence in cells in 96 wellplates was measured on fluorescent plate reader CytoFluor II (Beckman)at E_(ex)/E_(em) equal 480/520 nm and 540/575 nm for carboxyfluorescein-and carboxytetramethylrhodamine-modified substrates, respectively.Fluorescent images of the cells were taken on inverted epifluorescentmicroscope Axiovert-100 (Carl Zeiss) with filter sets appropriate fordetection of FITC and carboxytetramethylrhodamine.

Results

As shown in FIG. 16, CHO-K1 cells treated withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (25 μM, 5 minutes at 37° C.)could be quickly and efficiently loaded withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl. Image analysis indicated thatthe fluorescent dye crossed the cell membrane. FIG. 16 also shows thatcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl could be efficiently washed outof the cells. Taken together these data indicate that the plasmamembrane of CHO-K1 cells is permeable tocarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl.

In contrast, carboxyfluorescein-C₁₀H₂₁NO₂—Cl did not cross the plasmamembrane of CHO-K1 cells, even when cells were pretreated withcarboxyfluorescein-C₁₀H₂₁NO₂—Cl at high concentrations (i.e., 100 μM)and for much longer periods of time (60 minutes) (data not shown). Thus,the different permeabilities of the cell plasma membrane for varioussubstrates of the invention, e.g.,carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl andcarboxyfluorescein-C₁₀H₂₁NO₂—Cl, provides a unique opportunity to labelproteins expressed on the cell surface and proteins expressed inside thecell with different fluorophores, thereby allowing biplexing.

Example V DhaA-Based Tethering for Cell Imaging In Vivo

A. Colocalization of GFP and Carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl inLiving

Mammalian Cells

Materials and Methods

A GFP-connector-DhaA fusion cassette was constructed by replacing theRenilla luciferase coding region in Packard's vector codingGFP-DEVD-Rluc(h) (Packard #6310066) with DhaA.WT-Flag or DhaA.H272F-Flagcoding regions. Two primers

(5′-GGAATGGGCCCTCTAGAGCGACGATGTCA-3′; SEQ ID NO: 15, and5′-CAGTCAGTCACGATGGATCCGCTC AA-3′; SEQ ID NO: 16)were designed to add ApaI and BamHI sites (underlined) to the 5′ and 3′coding regions of DhaA, respectively, and to amplify a 980 bp fragmentfrom a pGEX-5X-3.DhaA.WT-Flag or pGEX-5X-3.DhaA.H272F-Flag template. TheR.Luc coding region was excised with ApaI and BamHI restriction enzymes.Then the 980 bp fragment containing DhaA was inserted into theApaI/BamHI site of the GFP-DEVD-Rluc(h) coding vector. The sequence ofthe gene fusion constructs was confirmed by DNA sequencing.

Cells transiently expressing GFP-connector-DhaA.WT-Flag orGFP-connector-DhaA.H272F-Flag fusion proteins were plated in LT-IIchambers (Nunc) at a density of 30,000 cells/cm². The next day, mediawas replaced with fresh media containing 25 μM ofcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl and the cells were placed backinto in a CO₂ incubator for 60 minutes. At the end of the incubation,media containing substrates was removed, cells were quickly washed withPBS (pH 7.4; four consecutive washes: 1.0 ml/cm²; 5 seconds each) andnew media was added to the cells. The cells were placed back into in aCO₂ incubator and after 60 minutes the cells were quickly washed withPBS (pH 7.4; four consecutive washes: 1.0 ml/cm²; 5 seconds each).Fluorescent images of the cells were taken on inverted epifluorescentmicroscope Axiovert-100 (Carl Zeiss) with filter sets appropriate fordetection of GFP and carboxytetramethylrhodamine.

Results

As shown by the images in FIG. 17, cells transfected with eitherGFP-connector-DhaA.WT-Flag or GFP-connector-DhaA.H272F-Flag showedrobust expression of the protein(s) with light emitting characteristicsof GFP. Analysis of the images of the same cells taken with acarboxytetramethylrhodamine-filter set showed that cells expressingGFP-connector-DhaA.WT-Flag were dark and could not be distinguished fromcells that do not express this fusion protein. In contrast, cellsexpressing GFP-connector-DhaA.H272F-Flag were very bright andunmistakably recognizable.

Western blot analysis of proteins isolated from CHO-K1 cells transfectedwith GFP-connector-DhaA.WT-Flag or GFP-connector-DhaA.H272F-Flag vectorsshowed that these cells expressed proteins that were recognized by ananti-Flag antibody and had the predicted molecular weight for the fusionproteins (data not shown). A fluoroscan of the SDS-PAGE gel with theseproteins showed strong/covalent binding of carboxytetramethylrhodamineto GFP-connector-DhaA.H272F-Flag and no binding toGFP-connector-DhaA.WT-Flag (FIG. 18).

B. Fusion Partners of DhaA in DhaA.WT-Flag and DhaA.H272F-Flag areFunctional

To determine whether fusion of two proteins leads to the loss of theactivity of one or both proteins, several DhaA-based fusion proteins(see Table II) with DhaA at the C- or N-terminus of the fusion and aconnector sequence, e.g., one having 13 to 17 amino acids, between thetwo proteins, were prepared. The data showed that the functionalactivity of both proteins in the fusion was preserved.

TABLE II Con- N-Terminal nec- C-terminal Function of Function of proteintor protein protein #1 protein #2 GST + DhaA.H272F Binding to bindingGSS column GFP + DhaA.H272F Green binding fluorescence R.Luc +DhaA.H272F hydrolysis of binding coelenterazine DhaA.H272F + R.LucBinding hydrolysis of coelenterazine DhaA.H272F + Flag bindingRecognized by antibodyC. Toxicity of Cl-AlkanesMaterials and Methods

To study the toxicity of Cl-alkanes, CHO-K1 cells were plated in 96 wellplates to a density of 5,000 cells per well. The next day, media wasreplaced with fresh media containing 0-100 μM concentrations ofCl-alkanes and the cells were placed back into a CO₂ incubator fordifferent periods of time. Viability of the cells was measured withCellTiter-Glo™ Luminescence Cell Viability Assay (Promega) according tothe manufacturer's protocol. Generally, 100 μl of CellTiter-Glo™ reagentwas added directly to the cells and the luminescence was recorded at 10minutes using a DYNEX MLX microtiter plate luminometer. In someexperiments, in order to prevent fluorescence/luminescence interference,the media containing fluorescent Cl-alkanes was removed and the cellswere quickly washed with PBS (pH 7.4; four consecutive washes: 1.0ml/cm²; 5 seconds each) before addition of CellTiter-Glo™ reagent.Control experiments indicated that this procedure had no effect on thesensitivity or accuracy of the CellTiter-Glo™ assay.

Results

As shown in FIG. 19, carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl showed notoxicity on CHO-K1 cells even after a 4 hour treatment at a 100 μMconcentration the (the highest concentration tested). After a 24 hourtreatment, no toxicity was detected at concentrations of 6.25 μM (the“maximum non-toxic concentration”). At concentrations>6.25 μM, therelative luminescence in CHO-K1 cells was reduced in a dose-dependentmanner with an IC₅₀ of about 100 μM. No toxicity of biotin-C₁₀H₂₁N₁O₂—Clwas observed even after 24 hours of treatment at 100 μM. In contrast,carboxy-X-rhodamine-C₁₀H₂₁NO₂—Cl had a pronounced toxic effect as areduction of the RLU in CHO-K1 cells could be detected after a 1 hourtreatment. The IC₅₀ value of this effect was about 75 μM with noapparent ATP reduction at a 25 μM concentration. The IC₅₀ value of5-carboxy-X-rhodamine-C₁₀H₂₁NO₂—Cl toxicity and the “maximum non-toxicconcentration” of 5-carboxy-X-rhodamine-C₁₀H₂₁NO₂—Cl decreased in atime-dependent manner reaching 12.5 μM and 6.25 μM, respectively.

D. Detection of DhaA.D106C in CHO Cells Contacted withCarboxytetramethylrhodamine- or DiAc-carboxyfluorescein-containingSubstrates and a Fixative

CHO cells (ATCC, passage 4) were seeded into 8-well chamber slides(German coverglass system) at low density in DMEM:F12 media (Gibco)containing 10% FBS and 1 mM glutamine (growth media) withoutantibiotics. Two days later, cells were inspected using an invertedphase microscope. Two visual criteria were confirmed before applying thetransfection reagents: 1) the level of cellular confluence per chamberwas approximately 60-80%, and 2)>90% of the cells were adherent andshowed a flattened morphology. The media was replaced with 150 μl offresh pre-warmed growth media and cells were incubated for approximately1 hour.

Cells were transfected using the TransIt TKO system (Miris). The TKOlipid was diluted by adding 7 μl of lipid per 100 μl of serum-freeDMEM:F12 media, and then 1.2 μg of transfection-grade DhaA.D106C DNA wasadded per 100 μl of lipid containing media. The mixture was incubated atroom temperature for 15 minutes, and then 25 μl aliquots weretransferred into individual culture chambers (0.3 μg DNA). Cells werereturned to the incubator for 5-6 hours, washed two times with growthmedia, 300 μl of fresh growth media was added, and then cells wereincubated for an additional 24 hours.

Transfected or non-transfected control cells were incubated with 12.5 μMcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl or 12.5 μMDiAc-carboxyfluorescein-C₁₀H₂₁NO₂—Cl in 10% FBS/DMEM for 30 minutes at37° C. and 5% CO₂. Cells were washed with warm growth media three times,300 μl fresh growth media was added, and then cells were incubated for 1hour.

Growth media was replaced with warm PBS and live cells were visualizedusing a Zeiss Axiovert 100 inverted microscope equipped with a rhodaminefilter set (Exciter filter=540, Emission filter=560LP) and a fluoresceinfilter set (Exciter filter=490, Emission filter=520), and a Spot CCDcamera. Images were captured with exposure times of 0.15-0.60 seconds atgain settings of 4 or 16.

Discreet and specifically labeled transfected cells were evident in bothcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl andDiAc-carboxyfluorescein-C₁₀H₂₁NO₂—Cl labeled cells. The majority ofcells were non-transfected cells and they did not retain the label.

The PBS was removed and cells were fixed with 3.7% paraformaldehyde/0.1%Triton in PBS for 15 minutes. The fixative was removed, PBS was added,and a second set of images was captured for bothcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl andDiAc-carboxyfluorescein-C₁₀H₂₁NO₂—Cl labeled cells.

The PBS was replaced with 50% methanol in PBS and cells were incubatedfor 15 minutes, followed by a 15 minute incubation in 95% methanol. Athird set of images was captured and then an equal volume mixture ofmethanol and acetone was applied to the cells and incubated for 15minutes. The media was replaced with PBS and a fourth set of images wascollected.

Results suggested that the binding of the substrates to the DhaA.D106Cmutant was stable following fixation with paraformaldehyde andsubsequent processing of fixed cell samples in methanol and acetone.Furthermore, the brightness of the carboxytetramethylrhodamine orcarboxyfluorescein fluorescence was unchanged under these conditions.

Example VI Mutant Beta-Lactamase (BlaZ)-Based Tethering

The serine-β-lactamases, enzymes that confer bacterial resistance toβ-lactam antibiotic, likely use the hydroxyl group of a serine residue(Ser70 in the class A consensus numbering scheme of Ambler et al.(1991)) to degrade a wide range of β-lactam compounds. The reactionbegins with the formation of a precovalent encounter complex (FIG. 20A),and moves through a high-energy acylation tetrahedral intermediate (FIG.20B) to form a transiently stable acyl-enzyme intermediate, forming anester through the catalytic residue Ser70 (FIG. 20C). Subsequently, theacyl-enzyme is attacked by hydrolytic water (FIG. 20D) to form ahigh-energy deacylation intermediate (FIG. 20E) (Minasov et al., 2002),which collapses to form the hydrolyzed product (FIG. 20F). The productis then expelled, regenerating free enzyme. As in serine proteases, thismechanism requires a catalytic base to activate the serine nucleophileto attack the amide bond of the substrate and, following formation ofthe acyl-enzyme intermediate, to activate the hydrolytic water forattack on the ester center of the adduct.

A. Mutant β-Lactamase and Fusions Thereof

Materials and Methods

The plasmid pTS32 harboring Staphylococcus aureus PC1 blaZ gene(Zawadzke et al., 1995) was kindly provided by Dr. O. Herzberg(University of Maryland Biotechnology Institute). The blaZ gene has thefollowing sequence:

(SEQ ID NO: 36) AGCTTACTAT GCCATTATTA ATAACTTAGC CATTTCAACA CCTTCTTTCAAATATTTATAATAAACTATT GACACCGATA TTACAATTGTAATATTATTG ATTTATAAAA ATTACAACTGTAATATCGGAGGGTTTATTT TGAAAAAGTT AATATTTTTAATTGTAATTG CTTTAGTTTTAAGTGCATGT AATTCAAACAGTTCACATGC CAAAGAGTTA AATGATTTAGAAAAAAAATATAATGCTCAT ATTGGTGTTT ATGCTTTAGATACTAAAAGT GGTAAGGAAG TAAAATTTAATTCAGATAAGAGATTTGCCT ATGCTTCAAC TTCAAAAGCG ATAAATAGTGCTATTTTGTTAGAACAAGTA CCTTATAATA AGTTAAATAAAAAAGTACAT ATTAACAAAG ATGATATAGTTGCTTATTCTCCTATTTTAG AAAAATATGT AGGAAAAGAT ATCACTTTAAAAGCACTTATTGAGGCTTCA ATGACATATA GTGATAATACAGCAAACAAT AAAATTATAA AAGAAATCGGTGGAATCAAAAAAGTTAAAC AACGTCTAAA AGAACTAGGA GATAAAGTAACAAATCCAGTTAGATATGAG ATAGAATTAA ATTACTATTCACCAAAGAGC AAAAAAGATA CTTCAACACCTGCTGCCTTCGGTAAGACCC TTAATAAACT TATCGCCAAT GGAAAATTAAGCAAAGAAAACAAAAAATTC TTACTTGATT TAATGTTAAATAATAAAAGC GGAGATACTT TAATTAAAGACGGTGTTCCAAAAGACTATA AGGTTGCTGA TAAAAGTGGT CAAGCAATAACATATGCTTCTAGAAATGAT GTTGCTTTTG TTTATCCTAAGGGCCAATCT GAACCTATTG TTTTAGTCATTTTTACGAATAAAGACAATA AAAGTGATAA GCCAAATGAT AAGTTGATAAGTGAAACCGCCAAGAGTGTA ATGAAGGAAT TTTAATATTCTAAATGCATA ATAAATACTG ATAACATCTTATATTTTGTA TTATATTTTG TATTATCGTT GAC.

GST-blaZ (WT and E166D, N170Q, or E166D:N170Q mutants) fusion cassetteswere constructed by introducing point mutations into the blaZ gene andcloning the blaZ coding regions into SalI/AgeI sites of pGEX5x3 vector.The internal mutagenic primers were as follows:

E166D (5′-CCAGTTAGATATGACATAGAATTAAATTACTATTCACC-3′, SEQ ID NO: 56;5′-GGTGAATAGTAATTTAATTCTATGTCATATCTAACTGG-3′, SEQ ID NO: 57); N170Q(5′-CCAGTTAGATATGAGATAGAATTACAGTACTATTCACC-3′, SEQ ID NO: 58; and5′-GGTGAATAGTACTGTAATTCTATCTCATATCTAACTGG-3′, SEQ ID NO: 59); andE166D:N170Q (5′CCAGTTAGATATGACATAGAATTACAGTACTATTCACC-3′; SEQ ID NO: 60and 5′-GGTGAATAGTACTGTAATTCTATGTCATATCTAACTGG-3; SEQ ID NO: 61).Two external primers

(5′-CAACAGGTCGACGCCGCCATGAAAGAGTTAAATGATTTAG-3′, SEQ ID NO: 62; and5′-GTAGTCACCGGTAAATTCCTTCATTACACTCTTGGC-3′, SEQ ID NO: 63)were designed to add N-terminal SalI site and a Kozak sequence to the 5′coding region, add an AgeI site to the 3′ coding regions of blaZ, and toamplify a 806 bp fragment from a blaZ.WT template. The resultingfragment was inserted into the SalI/AgeI site of the vector pGEX-5X-3containing a glutathione S-transferase (GST) gene, a sequence coding aFactor Xa cleavage site, and multiple cloning sites (MCS) followed by asequence coding for Flag and stop codons. These gene fusion constructswere confirmed by DNA sequencing.

The GST-BlaZ (WT or mutants) fusion proteins were overexpressed incompetent E. coli BL21 (λDE3) cells and purified essentially asdescribed for DhaA and GST-DhaA fusion proteins (except the potassiumphosphate buffer (0.1 M, pH 6.8) was used instead of Buffer A).Homogeneity of the proteins was verified by SDS-PAGE.

The chromogenic substrate 6-β-[(Furylacryloyl)amido]penicillanic acidtriethylamine salt (FAP) was purchased from Calbiochem (La Jolla,Calif.). Hydrolysis of FAP was monitored by loss of absorbance at 344 nm(deltaE=1330 M⁻¹ cm⁻¹) on a Beckman Du640 spectrophotometer (BeckmanCoulter, Fullerton, Calif.). All assays were performed at 25° C. in 0.1M potassium phosphate buffer at pH 6.8.

In CCF2, the cephalosporin core links a 7-hydroxycoumarin to afluorescein. In the intact molecule, excitation of the coumarin(E_(ex)—409 nm) results in FRET to the fluorescein, which emits greenlight (E_(em)—520 nm). Cleavage of CCF2 by β-lactamase results inspatial separation of the two dyes, disrupting FRET such that excitationof coumarin now gives rise to blue fluorescence (E_(ex)—447 nm). CCF2was purchased from Aurora Biosciences Corporation (San Diego, Calif.).Reduction of the FRET signal and an increase in blue fluorescence weremeasured on Fluorescence Multi-well Plate Reader CytoFluorII (PerSeptiveBiosystems, Framingham, Mass., USA).

Results

All β-lactamases, including β-lactamase from Staphylococcus aureus PC1,hydrolyze β-lactams of different chemical structure. The efficiency ofhydrolysis depends on the type of the enzyme and chemical structure ofthe substrate. Penicillin is considered to be a preferred substrate forβ-lactamase from Staphylococcus aureus PC1.

The effect of point mutation(s) on the ability of β-lactamase tohydrolyze penicillins was studied as described in Zawadzke et al.(1995). As shown in FIG. 20, a GST-β-lactamase PC1 fusion proteinefficiently hydrolyzed FAP. Hydrolysis of FAP by BlaZ.E166D, BlaZ.N170Qor BlaZ.E166D:N170Q BlaZ mutants could not be detected even after 60minutes of co-incubation. Therefore, these mutations lead to significantinactivation of BlaZ.

To show that BlaZ.E166D, BlaZ.N170Q, or BlaZ.E166D:N170Q mutants bindβ-lactams, and therefore different functional groups could be tetheredto these proteins via β-lactams, GST fusions of these mutants wereincubated with BOCELLIN™ FL, a fluorescent penicillin (Molecular ProbesInc., Eugene, Oreg.). Proteins were resolved on SDS-PAGE and analyzed onfluoroimager (Hitachi, Japan) at an E_(ex)/E_(em) appropriate for theparticular fluorophore. The data in FIG. 22 show that all BlaZ mutantsbind bocellin. Moreover, the bond between BlaZ mutants and fluorescentsubstrates was very strong, and probably covalent, since boiling withSDS followed by SDS-PAGE did not disrupt the bond. Also, the bindingefficiency of double mutant BlaZ.E166D:N170Q (judged by the strength ofthe fluorescent signal of protein-bound fluorophore) was much higherthan binding efficiency of either of the single mutants, and the bindingefficiency of BlaZ.N170Q was higher than binding efficiency ofBlaZ.E166D. These data, in combination with current understanding of therole of the individual amino acids in hydrolysis of beta-lactams, showthat additional mutations (e.g., a mutation of an auxiliary amino acid)can improve efficiency of tethering of functional groups to a mutatedprotein.

The effect of point mutation(s) on the ability of β-lactamase tohydrolyze cephalosporins was also studied using CCF2, a FRET-basedsubstrate described by Zlokarnik et al. (1998). As shown in FIG. 23, theGST-β-lactamase PC1 fusion protein efficiently hydrolyzed CCF2 (lane 2).Single point mutations (i.e., E166D or N170Q) reduced the ability of thefusion proteins to hydrolyze CCF2 (lanes 3 and 4). The replacement oftwo amino acids (BlaZ.E166D:N170Q mutants, lane 5) had an even morepronounced effect on the CCF2 hydrolysis. However, all BlaZ mutants werecapable of hydrolyzing CCF2.

Thus, an amino acid substitution at position 166 or 170, e.g., Glu166Aspor Asn170Gly enables the mutant beta-lactamase to trap a substrate andtherefore tether the functional group of the substrate to the mutantbeta-lactamase via a stable, e.g., covalent, bond. Moreover, mutation ofan amino acid that has an auxiliary effect on H₂O activation increasedthe efficiency of tethering.

Example VII Targeting of DhaA.H272F to the Nucleus and Cytosol of LivingCells

Materials and Methods

A GFP-connector-DhaA.H272F-NLS3 fusion cassette was constructed byinserting a sequence encoding NLS3 (three tandem repeats of the NuclearLocalization Sequence (NLS) from simian virus large T-antigen) into theAgeI/BamHI sites of a pCIneo.GFP-connector-DhaA.H272F-Flag vector. Twocomplementary oligonucleotides

(5′-CCGGTGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTATGAG-3′, sense, SEQ ID NO: 37, and5′-GATCCTCATACCTTTCTCTTCTTTTTTGGATCTACCTTTCTCTTCTTTTTTGGATCTACCTTTCTCTTCTTTTTTGGATCA-3′, antisense, SEQ ID NO: 38)coding for the NLS3 peptide, were annealed. The annealed DNA had an AgeIsite at 5′ end and a BamHI site at the 3′ end. The annealed DNA wassubcloned into the GFP-connector-DhaA.H272F-Flag construct at theAgeI/BamHI sites. The sequence of the gene fusion construct wasconfirmed by DNA sequencing.

A DhaA.H272F-β-arrestin2 fusion cassette was constructed by replacingthe pGFP² coding region in Packard's vector encoding GFP²-β-arrestin2(Packard #6310176-1F1) with the DhaA.H272F-Flag coding region. Twoprimers

(5′-ATTATGCTGAGTGATATCCC-3′; SEQ ID NO: 39, and5′-CTCGGTACCAAGCTCCTTGTAGTCA-3′; SEQ ID NO: 40)were designed to add a KpnI site to the 3′ coding region of DhaA, and toamplify a 930 bp fragment from a pGEX5X-3.DhaA.H272F-Flag template. ThepGFP² coding region was excised with NheI and KpnI restriction enzymes,then the 930 bp fragment containing encoding DhaA.H272F was insertedinto the NheI and KpnI sites of the GFP²-β-arrestin2 coding vector. Thesequence of the fusion construct was confirmed by DNA sequencing.

CHO-K1 or 3T3 cells transiently expressingGFP-connector-DhaA.H272F-NLS3, GFP²-β-arrestin2 orDhaA.H272F-β-arrestin2 fusion proteins were plated in LT-II chambers(Nunc) at a density of 30,000 cells/cm². The next day, media wasreplaced with fresh media containing 25 μM ofcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl and the cells were placed backinto a CO₂ incubator for 60 minutes. At the end of the incubation,substrate media was removed, cells were quickly washed with PBS (pH 7.4;four consecutive washes: 1.0 ml/cm²; 5 seconds each), and new media wasadded to the cells. The cells were placed back into a CO₂ incubator andafter 60 minutes the cells were quickly washed with PBS (pH 7.4; 1.0ml/cm²). Fluorescent images of the cells were taken on confocalmicroscope Pascal-5 (Carl Zeiss) with filter sets appropriate for thedetection of GFP and carboxytetramethylrhodamine.

Results

As shown by the images in FIG. 24, GFP and carboxytetramethylrhodaminewere co-localized in the cell nucleus of cells expressionGFP-connector-DhaA.H272F-NLS3 and contacted withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl.

As shown by the images in FIG. 25, GFP-β-arrestin2 expressing cells havea typical β-arrestin2 cytosolic localization. A fluoroscan of theSDS-PAGE gel of DhaA.H272F-β-arrestin2 showed strong binding of acarboxytetramethylrhodamine containing DhaA substrate to cellsexpressing DhaA.H272F-β-arrestin2.

Example VIII Site-Directed Mutagenesis of DhaA Catalytic Residue 130

Haloalkane dehalogenases use a three-step mechanism for cleavage of thecarbon-halogen bond (FIGS. 1A-B). This reaction is catalyzed by a triadof amino acid residues composed of a nucleophile, base and acid which,for the haloalkane dehalogenase from Xanthobacter autotrophicus (DhlA),are residues Asp124, His289 and Asp260, respectively (Franken et al.,1991), and in the Sphingomonas and Rhodococcus dehalogenase enzymes,LinB and DhaA, respectively, the analogous triad of residues have beenidentified as Asp108, His272 and Glu132 (Hynkova et al., 1999) andAsp106, His272 and Glu130 (Newman et al., 1999). After substratebinding, nucleophilic attack by the carboxylate of an Asp residue on thesubstrate causes the cleavage of the halogen-carbon bond and theformation of an alkyl-ester intermediate. Site-directed mutagenesisstudies on the DhlA Asp124 residue shows that this first reactionproceeds by covalent catalysis with the formation of an alkyl-enzymeintermediate (Pries et al., 1994). The next step in the dehalogenasereaction pathway is hydrolysis of the intermediate ester by a watermolecule activated by the active site His residue. While the catalytichistidine residue is the base catalyst for the dealkylation of thecovalent intermediate, it is not essential for the initial nucleophilicattack of the active site Asp. Protein mutants that lack the crucialcatalytic histidine residue have been shown to carry out the alkylationhalf reaction thereby producing a stable, covalent ester intermediate.For example, a His289Gln mutant of DhlA has previously been shown toaccumulate the covalent alkyl-enzyme intermediate (Pries et al., 1995).

Unlike the haloalkane dehalogenase nucleophile and base residues, therole of the third member of the catalytic triad is not yet fullyunderstood. The catalytic acid is hydrogen bonded to the catalytic Hisresidue and may assist the His residue in its function by increasing thebasicity of nitrogen in the imidazole ring. Krooshof et al. (1997),using site-directed mutagenesis to study the role of the DhlA catalyticacid Asp260, demonstrated that a D260N mutant was catalyticallyinactive. Furthermore, this residue apparently had an importantstructural role since the mutant protein accumulated mainly in inclusionbodies. The haloalkane dehalogenase from Sphinogomonas paucimobilis(LinB) is the enzyme involved in γ-hexachlorocyclohexane degradation(Nagata et al., 1997). Hynkova et al., (1999) replaced the putativecatalytic residue (Glu-132) of the LinB with glutamine (Q) residue.However, no activity was observed for the E132Q mutant even at very highsubstrate concentrations.

To examine the role of the DhaA catalytic triad acid Glu130 in proteinproduction and on the ability of the mutant protein to form covalentalkyl-enzyme intermediates with a fluorescent-labeled haloalkanesubstrate, site-directed mutagenesis was employed to replace the DhaAglutamate (E) residue at position 130 with glutamine, leucine andalanine

Materials and Methods

Strains and Plasmids.

Ultracompetent E. coli XL10 Gold (Stratagene; Tet^(r) Δ(mcrA)183Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F′proAB lacI ^(q)ZΔM15 Tn10 (Tet^(r)) Amy Cam^(r)]) was used to as a hostin transformation of site-directed mutagenesis reactions. E. coli strainJM109 (e14-(McrA) recA1 endA1 gyrA96 thi-1 hsdR17(rK− mK+) supE44 relA1Δ(lac-proAB) [F′ traD36 proAB lacI^(q)ZΔM15]) was used as the host forgene expression and whole cell enzyme labeling studies. A GST-DhaA-FLAGgene fusion cloned into plasmid pGEX5×3, designated pGEX5X3DhaAWT.FLAG,was used as the starting template for E130 mutagenesis. A mutant plasmidcontaining a H272F mutation in DhaA, designated pGEX5X3DhaAH272F-FLAG,was used as a positive control in labeling studies and the cloningvector pGEX5X3 was used as a negative control.

Site-Directed Mutagenesis of the DhaA E130 Residue.

The sequence of the oligonucleotides used for mutagenesis is shownbelow. The underlined nucleotides indicate the position of the alteredcodons. The oligonucleotides were synthesized by Integrated DNATechnologies (Coralville, Iowa) at the 100 nmole scale and modified byphosphorylation at the 5′ end.

DhaA E130Q (SEQ ID NO: 41) 5′CAAAGGTATTGCATGTATGCAGTTCATCCGGCCTATCCCG 3′ DhaA E130L (SEQ ID NO: 42)5′ GTCAAAGGTATTGCATGTATGCTGTTCATCCGGCCTATCCCGAC 3′ DhaA E130A(SEQ ID NO: 43) 5′ AGGTATTGCATGTATGGCGTTCATCCGGCCTATCCC 3′Site-directed mutagenesis was performed using the QuikChange Multi kitaccording to the manufacturer's instructions (Stratagene, La Jolla,Calif.). The mutagenesis reactions were introduced into competent E.coli XL10 Gold cells and transformants were selected on LB agar platescontaining ampicillin (100 μg/mL). Plasmid DNA isolated from individualtransformants was initially screened for the loss of an EcoRI site dueto replacement of the glutamate codon (GAAttc). Clones suspected ofcontaining the desired codon change from each reaction were selected andsubjected to DNA sequence analysis (SeqWright, Houston, Tex.). Theprimer used to confirm the sequence of the mutants in the pGEX5X3 vectorwas as follows: 5′GGGCTGGCAAGCCACGTTTGGTG 3′(SEQ ID NO:64).

DhaA Mutant Analysis.

The three.DhaA.E130 substitution mutants were compared to the followingconstructs: Wild-type DhaA, DhaA.H272F, and a DhaA negative control(pGEX5X3 vector only). Overnight cultures of each clone were grown in 2mL of LB containing ampicillin (100 μg/mL) by shaking at 30° C. Theovernight cultures were diluted 1:50 into a sterile flask containing 50mL fresh LB medium and ampicillin (100 μg/mL). The cultures wereincubated with shaking at 25° C. to minimize the production of insolubleprotein species. When the cultures reached mid-log phase (OD₆₀₀=0.6),IPTG (0.1 mM) was added and the cultures were incubated with shaking at25° C. for an additional 22 hours. For labeling of whole cells with acarboxytetramethylrhodamine haloalkane conjugated substrate, the celldensity of each culture was adjusted to OD₆₀₀=1 prior to addingsubstrate to a concentration of 15 μM. The cells were incubated withgentle agitation at 4° C. for approximately 18 hours. Followingincubation, 20 μl of cells from each labeling reaction was added to 6 μlof 4×SDS loading dye and the samples were boiled for about 3 minutesprior to being loaded onto a 4-20% acrylamide gel (Tris glycine). For invitro labeling studies, crude lysates of IPTG induced cultures wereprepared by collecting 3 mL of cells (OD₆₀₀=1) and resuspending theresulting pellet in 75 μL PBS. Following a freeze/thaw step, 225 μL of1× Cell Culture Lysis Reagent (Promega Corp., Madison, Wis.) containing1.25 mg/mL lysozyme was added to facilitate lysis of the cells. A 20 μLsample of each lysate was combined with 25 μL of 1×PBS. Thecarboxytetramethylrhodamine labeled haloalkane substrate was added to afinal concentration of 25 μM. The labeling reactions were incubated atroom temperature for 2 hours. A 25 μl sample of each labeling reactionwas added to 6 μl of 4×SDS loading dye and the samples were boiled forabout 3 minutes prior to being loaded onto a 4-20% acrylamide gel (Trisglycine). The gels were imaged using a FluorImager SI instrument(Amersham Biosciences, Piscataway, N.J.) set to detect emission at 570nm.

Cell-free lysates were generated by centrifugation of crude lysates for15 minutes at 14,000 RPM. Protein production was monitored by SDS-PAGEand Western blot analysis. Proteins transferred to a PVDF membrane wereincubated with an anti-FLAG® antibody conjugated with alkalinephosphatase (AP) (Sigma, St. Louis, Mo.). The blot was developed withthe Western Blue stabilized substrate for alkaline phosphatase (PromegaCorp., Madison, Wis.).

Results

The role of the DhaA catalytic acid in the hydrolysis of thealkyl-enzyme intermediate was probed by site-directed mutagenesis. TheDhaA.WT codon E130 was replaced with a codon for glutamine (Q), leucine(L) or alanine (A), as these substitutions would likely be leastdisruptive to the structure of the enzyme. Following mutagenesis,restriction endonuclease screening and DNA sequence analysis was used toverify the desired codon changes. Sequence verified DhaA.E130Q,DhaA.E130L and DhaA.E130A clones, designated C1, A5 and A12,respectively, were chosen for further analysis. The E130 mutants wereanalyzed for protein expression and for their ability to form a covalentalkyl-enzyme intermediate with a carboxytetramethylrhodamine labeledhaloalkane substrate. The three E130 gene variants were over-expressedin E. coli JM109 cells following induction with IPTG. SDS-PAGE analysisof crude cell lysates showed that cultures expressing the wild-type andmutant dhaA genes accumulated protein to approximately the same level(FIG. 26; lanes 2, 4, 6, 8, 10, and 12). Furthermore, the protein thatwas produced by constructs encoding DhaA.WT and DhaA.H272F was for themost part soluble since the amount of protein did not change appreciablyafter centrifugation (FIG. 26; lanes 3 and 5). The abundant 22 kDaprotein bands present in the vector only lanes (FIG. 26; lanes 6 and 7)represented the GST protein. These results, however, are in starkcontrast to the DhaA.E130Q, DhaA.E130L and DhaA.E130A mutants thatappeared to accumulate predominantly insoluble DhaA protein. Thisconclusion is based on the observation that after centrifugation, therewas a significant loss in the amount of DhaA protein present incell-free lysates (FIG. 26; lanes 9, 11, and 13). Nevertheless, aprotein band that comigrates with DhaA was clearly observed in eachDhaA.E130 mutant lanes after centrifugation (+s) suggesting the presenceof soluble enzyme. Western analysis was, therefore, used to determine ifthe protein bands observed in the DhaA.E130 mutants followingcentrifugation represented soluble DhaA material. The immunoblot shownin FIG. 27 confirmed the presence of soluble DhaA protein in each of theDhaA.E130 mutant cell-free lysates (lanes 9, 11, and 13).

The DhaA.E130 mutants were also examined for their ability to generatean alkyl-enzyme covalent intermediate. Crude lysates prepared from IPTGinduced cultures of the various constructs were incubated in thepresence of the carboxytetramethylrhodamine labeled substrate. FIG. 28showed that the DhaA.H272F mutant (lane 3) was very efficient atproducing this intermediate. No such product could be detected witheither the DhaA.WT or negative control lysates. Upon initialexamination, the DhaA.E130 mutants did not appear to produce detectablelevels of the covalent product. However, upon closer inspection of thefluoroimage extremely faint bands were observed that could potentiallyrepresent minute amounts of the covalent intermediate (FIG. 28; lanes5-7). Based on these results, the ability of whole cells to generate acovalent, fluorescent alkyl-enzyme intermediate was investigated.

FIG. 29 shows the results of an in vivo labeling experiment comparingeach of the DhaA.E130 mutants with positive (DhaA.H272F mutant) andnegative (DhaA-) controls. As expected, the DhaA.H272F mutant wascapable of generating a covalent alkyl-enzyme intermediate as evidencedby the single fluorescent band near the molecular weight predicted forthe DhaA fusion (FIG. 29, lane 3). As previously observed with the invitro labeling results, no such product could be detected with eitherthe wild-type or negative control cultures (FIG. 29, lanes 2 and 3) butvery faint fluorescent bands migrating at the correct position wereagain detected with all three DhaA.E130 substituted mutants (FIG. 29,lanes 5-7). These results point to the possibility that the DhaA.E130Q,L and A mutants have the ability to trap covalent alkyl-enzymeintermediates. The efficiency of this reaction, however, appears toproceed at a dramatically reduced rate compared to the DhaA.H272F mutantenzyme.

The results of this mutagenesis study suggest that the DhaA catalyticacid residue DhaA.E130 plays an important structural role in the correctfolding of the enzyme. The DhaA protein was clearly sensitive tosubstitutions at this amino acid position as evidenced by the presenceof largely insoluble protein complexes in the DhaA.E130Q, DhaA.E130L andDhaA.E130A crude lysates. Nevertheless, based on SDS-PAGE and immunoblotanalyses, a significant quantity of soluble DhaA protein was detected inthe cell-free lysates of all three DhaA.E130 mutants.

Example IX Capturing of DhaA.H272F-Flag and DhaA.H272F-Flag RenillaLuciferase Fusion Proteins Expressed in Living Mammalian Cells

Materials and Methods

CHO-K1 cells were plated in 24 well plates (Labsystems) at a density of30,000 cells/cm² and transfected with a pCIneo.DhaA.WT-Flag orpCIneo.hRLuc-connector-DhaA.H272F-Flag vector. Twenty-four hours later,media was replaced with fresh media containing 25 μMbiotin-C₁₀H₂₁N₁O₂—Cl and 0.1% DMSO, or 0.1% DMSO alone, and the cellswere placed in a CO₂ incubator for 60 minutes. At the end of theincubation, the media was removed, cells were quickly washed with PBS(pH 7.4; four consecutive washes; 1.0 ml/cm²; 5 seconds each) and newmedia was added to the cells. In some experiments, the media was notchanged. The cells were placed back in a CO₂ incubator.

After 60 minutes, media was removed, and the cells were collected in PBS(pH=7.4, 200 μl/well, RT) containing protease inhibitors (Sigma #P8340).The cells were lysed by trituriation through a needle (IM1 23GTW). Then,cell lysates were incubated with MagnaBind Streptavidin coated beads(Pierce #21344) according to the manufacturer's protocol. Briefly, celllysates were incubated with beads for 60 minutes at room temperature(RT) using a rotating disk. Unbound material was collected; beads werewashed with PBS (3×500 μl, pH=7.4, RT) and resuspended in SDS-samplebuffer (for SDS-PAGE analysis) or PBS (pH=7.4, for determination ofR.Luc activity). Proteins were resolved on SDS-PAGE, transferred to anitrocellulose membrane, analyzed with anti-Flag-Ab or anti-R.Luc-Ab,and bound antibody detected by an enhanced chemiluminescence (ECL)system (Pharmacia-Amersham). Activity of hR.Luc bound to beads wasdetermined using Promega's “Renilla Luciferase Assay System” accordingto the manufacturer's protocol.

Results

Capturing of proteins expressed in living cells allows for analysis ofthose proteins with a variety of analytic methods/techniques. A numberof capturing tools are available although most of those tools requiregeneration of a highly specific antibody or genetically fusing a proteinof interest with specific tag peptides/proteins (Jarvik and Telmer,1998; Ragaut et al., 1999). However, those tags have only limited usefor live cell imaging. To capture DhaA.H272F and functional proteinsfused to DhaA.H272F, SA-coated beads were used (Savage et al., 1992).

Biotin-C₁₀H₂₁NO₂—Cl was efficiently hydrolyzed by DhaA.WT, andcovalently bound to DhaA.H272F and DhaA.H272F fusion proteins in vitroand in vivo. Moreover, binding was observed both in E. coli and inmammalian cells. Control experiments indicated that about 80% of theDhaA.H272F-Flag protein expressed in CHO-K1 cells was labeled after a 60minute treatment.

CHO-K1 cells transiently expressing DhaA.H272F-Flag were treated withbiotin-C₁₀H₂₁NO₂—Cl. Biotin-C₁₀H₂₁NO₂—Cl treated cells were lysed andcell lysates were incubated with SA-coated beads. Binding of DhaA.H272Fto beads was analyzed by Western blot using anti-Flag® antibody. Asshown in FIG. 30D, DhaA.H272F-Flag capturing was not detected in theabsence of biotin-C₁₀H₂₁NO₂—Cl treatment. At the same time, more than50% of the DhaA.H272F-Flag expressed in cells was captured on SA-coatedbeads if the cells were treated with biotin-C₁₀H₂₁NO₂—Cl.

To show the capturing of functionally active proteins fused toDhaA.H272F-Flag, cells were transfected with a vector encodinghR.Luc-connector-DhaA.H272F-Flag, and the luciferase activity capturedon the beads measured. As shown in FIG. 30C, significant luciferaseactivity was detected on beads incubated with a lysate ofbiotin-C₁₀H₂₁NO₂—Cl treated cells. At the same time, no luciferaseactivity was detected on beads incubated with a lysate from cells thatwere not treated with biotin-C₁₀H₂₁NO₂—Cl. Moreover, no hR.Luc activitywas detected on beads incubated with lysate from the cells treated withbiotin-C₁₀H₂₁NO₂—Cl when free biotin-C₁₀H₂₁NO₂—Cl was not washed out.

Taken together, these data show that functionally active protein(hR.Luc) fused to the DhaA.H272F can be efficiently captured usingbiotin-C₁₀H₂₁NO₂—Cl and SA-coated beads. The capture isbiotin-dependent, and can be competed-off by excess ofbiotin-C₁₀H₂₁NO₂—Cl. As a significant inhibitory effect of the beads onthe hR.Luc activity was observed (data not shown), SDS-PAGE and Westernblot analysis with anti-R.Luc antibody were used to estimate theefficiency of capture of hR.Luc-connector-DhaA.H272F-Flag fusionprotein. As shown in FIG. 30D, more than 50% ofhR.Luc-connector-DhaA.H272F-Flag fusion protein can be captured inbiotin-dependent manner. This is in good agreement with the capturingefficiency of DhaA.H272F-Flag (see FIG. 30A).

Example X DhaA Mutants with Increased Rates of Covalent Bond Formation

Replacement of the DhaA catalytic base His272 with a phenylalanineresidue is compatible with the Asp nucleophile and resulted in amodified protein, designated DhaA.H272F, that accumulates substantialamounts of the covalent alkyl-enzyme intermediate (FIG. 2C). The absenceof the water activating His272 residue allows trapping of the covalentester intermediate (FIG. 2C). A structural model of such a mutant beforebinding and after binding a substrate is shown in FIGS. 2E and 2Frespectively. Furthermore, a DhaA mutant containing a cysteinesubstitution for the nucleophile residue Asp106 was also capable oftrapping covalent intermediates. This mutant, designated DhaA.D106C(FIG. 2D), displaces the halide moiety through the action of a thiolatenucleophile. The resulting thioether bond is stable to hydrolysis evenin the presence of the water activating H272 residue (FIG. 2D).

The ability to generate a stable, covalent linkage between protein andhaloalkane ligand provides for a universal reporter technology which cansite-specifically label, localize, immobilize and/or fluorescentlyvisualize proteins in mammalian cells (see Examples II-IX). In oneexample, active-site mutants of dehalogenase (DhaA) tether fusionproteins with those mutants via a stable, covalent bond to synthetichaloalkane conjugated substrates. To enhance the kinetics of DhaA.H272Fand DhaA.D106C, modeling of protein-ligand (protein-substrate) complexeswas employed in an effort to identify favorable interactions betweenDhaA and a substrate so as to optimize the rate of covalent bondformation.

Materials and Methods

Strains, Growth Conditions and Plasmids.

E. coli strains DH10B (F-mcrA Δ[mrr-hsdRMS-mcrBC] φ80lacZΔM15 ΔlacX74deoR recA1 endA1 araD139 Δ(ara, leu)7697 galU galK rpsL nupG) and JM109(e14-(McrA) recA1 endA1 gyrA96 thi-1 hsdR17(r_(K)-m_(K)+) supE44 relA1Δ(lac-proAB) [F′ traD36 proAB lacI^(q)ZΔM15]) were used as the hosts forgene expression and for library screening. E. coli was routinely grownin Luria-Bertani (LB) or Terrific broth (TB) media (Sambrook et al.,2001). When required, Difco agar was added to the medium at 1.5% (w/v).Ampicillin (100 μg/mL; Amp) was added to the medium to select forrecombinant plasmids. The E. coli expression plasmidspGEX5X3DhaA.H272F.FLAG and pGEX5X3DhaA.D106C.FLAG containing GST fusionsto DhaA.H272F and DhaA.D106C, respectively, were used as the startingtemplates for site-directed mutagenesis. The expression vector pCI-Neo(Promega Corporation, Madison, Wis.) was used to examine expression andlabeling of DhaA mutants in mammalian cells.

Reagents and Chemicals.

All chemicals were purchased from Sigma-Aldrich (Milwaukee, Wis.). Allenzymes were from Promega (Madison, Wis.) unless otherwise noted. Themutagenesis and PCR primers were synthesized by Promega Corp., SeqWright(Houston, Tex.) and Integrated DNA Technologies (Coralville, Iowa).Mutagenesis of DhaA was performed using the QuikChange Multi kit(Stratagene, La Jolla, Calif.).Carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl,Carboxyfluorescein-C₁₀H₂₁NO₂—Cl, diacetylcarboxyfluorescein-C₁₀H₂₁NO₂—Cl, and biotin-containing chlorohaloalkaneligands (e.g., biotin-14-Cl, biotin-X-14-Cl, and biotin-PEG4-14-Cl, seeFIG. 7) were synthesized by Promega Biosciences Inc. (San Luis Abispo,Calif.).

DNA Analysis and Protein Modeling.

DNA analysis was performed using Vector NTI software package, version 8.Protein structures were obtained from the Protein Data Bank (PDBhttp://www.rcsb.org/pdb/). Structural analyses and modeling wereperformed with InsightII 2000.1 including modules Biopolymer, Discover,Homology, and Modeler (Accelrys http://www.accelrys.com/).

Mutagenesis and Library Construction.

Recombinant DNA work was performed using standard protocols as describedby Sambrook et al. (2001). Prior to mutagenesis, the sequence of DhaAtemplates was confirmed using the following oligonucleotides: forwardprimer “21972”,

forward primer “21972”, 5′-GGGCTGGCAAGCCACGTTTGGTG-3′ (SEQ ID NO: 64)and reverse primer “21973”, 5′-CCGGGAGCTGCATGTGTCAGAGG-3′(SEQ ID NO: 65).

The sequence of the oligonucleotides used for site-saturationmutagenesis of DhaA.H272F or DhaA.D106C residues 175 (Lys), 175 (Cys),and 273(Tyr) are shown below:

175 NNK:

(SEQ ID NO: 66) 5′ATCGAGGGTGCGCTCCCGNNKTGCGTCGTCCGTCCGCTTACGG 3′176 NNK:

(SEQ ID NO: 67) 5′ATCGAGGGTGCGCTCCCGAAANNKGTCGTCCGTCCGCTTACGG 3′175/176 NNK/NNK:

(SEQ ID NO: 68) 5′ATCGAGGGTGCGCTCCCGNNKNNKGTCGTCCGTCCGCTTACGG 3′Y273 NNK=H272F:

(SEQ ID NO: 69) 5′ATCGGCCCGGGATTGTTCNNKCTCCAGGAAGACAACCCGG 3′Y273 NNK=H272:

(SEQ ID NO: 70) 5′CGGCCCGGGATTGCACNNKCTCCAGGAAGACAACCCGGA 3′V245T:

(SEQ ID NO: 83) 5′GGGCACACCCGGCACCCTGATCCCCCCGG 3′The underlined nucleotides indicate the position of the altered codons.Site-directed mutagenesis was performed using the QuikChange Multi kitaccording to the manufacturer's instructions (Stratagene, La Jolla,Calif.). The mutagenesis reactions were introduced into competent E.coli and transformants were selected on LB agar plates containing Amp(100 μg/mL). Library quality was evaluated by DNA sequence analysis of12-48 randomly selected clones from each library. Plasmids for sequenceanalysis were isolated from E. coli using Wizard SV Miniprep Kits(Promega Corp.). DNA sequence analysis was performed by SeqWright DNATechnology Services (Houston, Tex.).

Sequencing primers for analyzing the 175, 176 and 175/176 librariesincluded:

“175/176”, (SEQ ID NO: 71) 5′-GCCTATCCCGACGTGGGACG-3′; “255R”,(SEQ ID NO: 72) 5′-AGGTCTCGCGGCTTCGGCCGGGGG-3′; “F70”, (SEQ ID NO: 73)5′-AAAATCGGACAAACCAGACCTCG-3′; “F189”,  (SEQ ID NO: 74)5′-ATCGCGAGCCCTTCCTCAAGCCTG-3′; and “R121”,  (SEQ ID NO: 75)5′-GTTCCGGATTGCGCTTGGCCCAGT-3′.Screening Assay Development.

In Vivo Detection of Binding to DhaA Substrates.

E. coli colonies harboring DhaA.H272F or DhaA.D106C encoding plasmidswere inoculated into 200 μL LB+100 μg/ml Amp and grown over night at 37°C. in flat bottom 96 wells plates. Overnight cultures were diluted 1:20into 200 μL TB+100 μg/mL Amp+0.1 mM IPTG and grown overnight at 30° C.The volume of cells used for in vivo labeling was normalized to growth(OD₆₀₀). 50 to 100 μL of induced cells were transferred to a U-bottom 96well plate, pelleted, re-suspended with 50 μl PBS+15 μMcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl and labeled at room temperaturefor 60 minutes on a rotating shaker. To remove the unbound ligand, cellswere harvested at 2500 rpm for 5 minutes, supernatants were discardedand the cells were re-suspended with 100 μl of 10 mM Tris-HCl pH 7.5,0.9% NaCl and 0.05% Triton and washed for 15 minutes. This washingprocedure was repeated 3 times. Fluorescence intensity was measured on aTecan Safire plate reader using the following parameters: 545 nmexcitation; 575 nm emission. The fluorescence intensity of DhaA mutantswas compared to DhaA-, DhaA.H272F and DhaA.D106C control cells.

Substrate Capture Using Immobilized DhaA.

Purified DhaA.H272F or DhaA.D106C mutant proteins (purified, 50 ng fromE. coli lysates generated using FastBreak™ cell lysis reagent, PromegaCorp.) was immobilized using 96-well microtiter plates (flat bottom;Nunc MaxiSorp) previously coated with anti-Flag M2 IgG (Sigma). Coatingtook place overnight at 4° C. using 100 μL, anti-Flag (5 μg/mL) in 0.1 MNaHCO₃ pH 9.6. The next day plates were emptied and blocked with 300 μL,PBS containing 3% BSA for 1 hour at 25° C. Plates were emptied andwashed 4× with PBS containing 0.1% Tween 20 (PBST), and biotinylatedsubstrate (varying concentrations of biotin-14-Cl, biotin-X-14-Cl, orbiotin-PEG4-14-Cl) was added to the wells in 100 μL, of PBS+0.05% Tween20+0.5% BSA (PBSTB) and incubated for various times at 25° C. Reactionsbetween immobilized DhaA and substrate were stopped by emptying platesand washing 4× with PBST. 100 μL, Streptavidin (SA)-HRP (1:5,000 inPBSTB; Prozyme) was then added to the wells and incubated for 1 hour at25° C. The plates were emptied and washed 8× with PBST, and TMB wasadded in a volume of 100 μL. After 15 minutes, color development wasstopped by the addition of an equal volume of 0.2 M H₂SO₄ and signalswere quantitated by measuring the absorbance at 450 nm.

Protein Capture Using MagneGST™ Paramagnetic Particles (PMPs).

Bacterial colonies were picked into 96-well plates containing LB+Amp andincubated with shaking at 30° C. The cultures were diluted 1:20 into 96well plates containing fresh TB medium, Amp and 0.1 mM IPTG. The plateswere incubated at 30° C. with shaking overnight. The 96 well platescontaining the IPTG induced cultures were centrifuged and supernatantsremoved. DhaA mutants were normalized for protein concentration bysaturating protein capture on MagneGST™ PMPs. A cocktail containingMagneGST™ cell lysis reagent, MagneGST™ PMPs andcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (15 μM) was pipetted into the96 well plates containing the cell pellets. The plates were shaken atabout 900 rpm for 10 minutes at room temperature. The particles werewashed three times with PBST using a MagnaBot® 96 magnetic separationdevice. The wash solution was removed and MagneGST™ elution solution wasadded and the plates were allowed to shake at room temperature (about900 rpm for 5 minutes). Supernatants were transferred to a new, flatbottom, transparent 96 well plate and the fluorescence intensity wasmeasured using an excitation wavelength at 550 nm and an emissionwavelength at 580 nm.

Automated Library Screening.

The DhaA mutant libraries were screened with the MagneGST™ based assayon a custom Tecan Freedom robotic workstation. The assay parameters wereautomated using the FACTS scheduling software and allowed the processingof multiple 96 well plates in parallel. The cell pellets were stored ina refrigerated Storex incubator (4° C.) until the plates wereautomatically retrieved for further processing. Reagents weretransferred to plates using a TeMo liquid handling system (Tecan US).The plates were shaken at about 900 rpm for 10 minutes on TecanTe-Shake™ at room temperature. The particles were washed with PBST usinga MagnaBot® 96 magnetic separation devices that were adapted to be usedon a TeMo liquid dispensing system and compatible with the FACTSscheduling software. Fluorescence intensity measurements were performedusing a Tecan Safire spectrofluorometer. Raw fluorescence intensity datawere imported into an Excel spreadsheet for analysis. The screening datawas examined for wells with higher intensity than the parental controlsindicating the potential presence of improved DhaA clones.

Secondary Library Screening.

Following the initial screening, all clones showing at least 20%improvement over parental clones (i.e., DhaA.H272F or DhaA.D106C) werestreaked onto LB plates supplemented with Amp. Four colonies at randomof each identified hit were inoculated into 200 μL LB+Amp and grownovernight at 30° C. in flat bottom 96 well plates. Overnight cultureswere diluted 1:50 into 200 μL TB ampicillin supplemented with 0.1 mMIPTG and grown overnight at 30° C. and 37° C. Induced cultures werere-assayed using the MagneGST™ based screen. All improved clones weresequenced and archived. Qiagen mini prep kit was used to prepare plasmidDNAs of sequencing. 2 ml cultures of all improved clones were archivedat −70° C. in the presence of 1% DMSO.

DhaA Protein Purification.

Proteins were purified on a small scale using the MagneGST™ proteinpurification system (Promega, Madison, Wis.). For protein purification,colonies were inoculated into 1 ml LB+100 μg/ml Amp and grown overnightat 30° C. Overnight cultures were diluted 1:50 into 10 mL of freshLB+100 μg/mL Amp. These cultures were grown until A₆₀₀=0.6 at whichpoint the cultures were induced with 0.1 mM IPTG and grown overnight at25° C. The cell pellets of induced cultures were frozen at −70° C. for15 minutes. To generate cell lysates, pellets were resuspended with 2 mLlysis buffer (containing 1 mM DTT+20 μL RQ DNase in the presence of 1×protease inhibitor cocktail (Becton-Dickinson Biosciences) and incubatedon a rotating shaker for 30 minutes. Four mLs of a 25% slurry ofMagneGST particles were equilibrated 3 times with the MagneGSTbinding/wash buffer prior to use. Following the final wash, theparticles were resuspended in 1× volume of the binding/wash buffer. Theparticles were added directly to the lysate and the mixture wasincubated for 30 minutes at room temperature on a rotating shaker toallow binding of the GST-DhaA fusion protein to the magnetic particles.Following washing of the particles 3 times with 2.5× volumes of washingbinding buffer+1 mM DTT, the GST-DhaA protein was eluted by incubationfor 15 minute with elution buffer (100 mM glutathione, 50 mM Tris HCl,pH 8.1, 1 mM DTT and 1×BD protease inhibition cocktail). The elutedprotein was dialyzed twice against storage buffer (50 mM Tris HCl, pH7.5, 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 20% glycerol).

Large-scale purification of DhaA protein fusions was accomplished usingGlutathione Sepharose 4 Fast Flow resin (Amersham Biosciences). Briefly,the pellet from a 500 mL culture of induced cells was resuspended in 20mL of 1× phosphate buffered saline (PBS) containing 1 mM DTT (buffer A).Following the addition of lysozyme (10 mg/mL), the mixture was allowedto incubate at 4° C. for 30 minutes. The protease inhibitor PMSF wasadded to a final concentration of 2 mM just prior to sonication. Clearedlysates were added to the resin and incubated with mixing 2 hours toovernight at 4° C. Following two 40 mL batch washes with buffer A, theresin was added to a Wizard Maxi column (Promega Corp.). The columncontents were washed 2× with 10 mL buffer A containing 0.3 M NaCl. Thefusion protein was eluted in 2 mL fractions of buffer A containing 20 mMglutathione. The protein containing fractions were dialyzed twice with 1L buffer A containing 20% glycerol.

In Vitro Labeling of Purified DhaA Mutants.

Covalent tethering of fluorescent substrates to DhaA mutants wasdetected by fluorimage gel analysis. GST-DhaA mutants (9 nM) wereincubated with carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl,carboxyfluorescein-C₁₀H₂₁NO₂—Cl, or rhodamine green-C₁₀H₂₁NO₂—Cl atvarious concentrations and temperatures in 50 mM Tris HCl (pH 7.5).Reactions were initiated by the addition of substrate, and for timecourse experiments 18 μL aliquots of the reactions were removed to tubescontaining 6 μL SDS gel loading buffer, boiled for 5 minutes, andresolved on pre-poured, 4-20% gradient SDS-polyacrylamide gels inTris-glycine (Invitrogen, Carlsbad, Calif.). Gels were fluorimaged usinga Hitachi FM Bio II (535 nm excitation, 580 nm emission) and bandsquantitated by either densitometry or ImageQuant (Amersham). Rateconstants were calculated from the following second-order rate equation(Cornish-Bowden, 1995):kt=(1/B ₀ −A ₀)ln [(B ₀ −x)A ₀/(A ₀ −x)B ₀]where k=the rate constant; B₀=[reactant B] at time=0, mol/L (M);A₀=[reactant A] at time=0, mol/L (M); B₀−x=[reactant B] at time=t, mol/L(M); and A₀−x=[reactant A] at time=t, mol/L (M). A plot of ln[(B₀−x)A₀/(A₀−x)B₀] versus time should be linear, and k can bedetermined from the slope of the line, k (B₀−A₀).

Fluorescence Polarization (FP).

Fluorescent polarization was used to analyze the reaction kinetics ofDhaA mutants. Measurements were taken on the Beacon 2000 (Invitrogen,Carlsbad, Calif.) or in a 96 well format using the Ultra plate reader(Tecan, Research Triangle Park, N.C.).Carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl orcarboxyfluorescein-C₁₀H₂₁NO₂—Cl substrates (7.5-10 nM) were incubatedwith an excess of purified GST-DhaA proteins. Forcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl labeling studies the followingconcentrations of protein were used: parental protein, 15 μM; firstgeneration DhaA mutants, 1.5-0.15 μM; and second generation DhaAmutants, 0.035 μM. For carboxyfluorescein-C₁₀H₂₁NO₂—Cl labeling studiesthe following concentrations of protein were used: parental protein, 15μM, first generation clones, 1.5-0.15 μM, and second generation clones,0.15 μM. Reactions were started by addition of protein to thesubstrates. Measurements of fluorescent polarization and fluorescentintensity were taken in 10-30 second intervals for 0.5-12 hours. Rateconstants were calculated using the 2^(nd) order rate equation.

Thermostability Analysis.

The thermostability profiles of the DhaA mutants were determined bymeasuring the residual activity of the purified proteins following 15,30 or 60 minute incubations at 4, 22, 30, 37, 42, 50 or 60° C. The FPassay was performed at room temperature (about 25° C.). For thesestudies, 15 μM parental or 1.5-0.15 μM of 1^(st) generation clones werelabeled with carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl and 0.15 nM of2^(nd) generation clones were labeled withcarboxyfluorescein-C₁₀H₂₁NO₂—Cl. For each clone, the labeling rate(slope of the linear range) was calculated for each condition. The rateobserved following a 15 minute incubation at 4° C. was arbitrarilyassigned as 100% activity. The residual activity (%) was calculated foreach condition. To determine stability, for each incubation time, the %of residual activity was plotted against the incubation temperatures. Tocalculate half-life, for each incubation temperature, the % of residualactivity was plotted against the incubation time. The time where 50%activity was lost was extrapolated from the graph.

Use of Immobilized DhaA to Capture Chloroalkylated Molecules.

Mutant DhaA (50 ng) was immobilized as above using microtiter platescoated with anti-Flag M2 IgG. Varying concentrations of chloroalkanewere incubated at 25° C. with model molecules of interest in solution(PBSTB). In the case of biotinylated chloroalkanes, the molecule ofinterest was SA-HRP. In the case ofcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl, the molecule of interest wasan anti-TMR IgG (Probes). The chloroalkylation reactions proceeded for 1hour and were then added (in a volume of 100 μl) to washed platescontaining immobilized DhaA. These incubations lasted for 1 to 2 hoursat 25° C., and were stopped by emptying the plates and washing 4× withPBST. For the SA-HRP reactions, TMB was added to the plates in a volumeof 100 μL. Color was developed for 15 minutes and then stopped by theaddition of an equal volume of 0.2 M H₂SO₄. Signals were quantitated bymeasuring absorbance at 450 nm. For the carboxytetramethylrhodaminereactions, a secondary anti-rabbit IgG-HRP conjugate (100 μL of a1:5,000 dilution in PBSTB; 1 hour at 25° C.) was used to detect boundanti-carboxytetramethylrhodamine IgG. Plates were washed 8× with PBST,developed with TMB, and quantitated as above.

DhaA Capture Using Immobilized Chloroalkane Substrates.

Biotinylated chloroalkane substrates, biotin-14-Cl, biotin-X-14-Cl, andbiotin-PEG4-14-Cl, were immobilized using streptavidin high bindingcapacity coated 96 well microtiter plates (flat bottom, Pierce). Usingan excess of substrate (about 2 μmol), the plates could bindapproximately 75 μmol of biotin per well. Following immobilization ofsubstrate for 1 hour at 25° C. in a buffer containing 100 μL PBS+0.05%Tween 20+0.5% BSA (PBSTB), plates were emptied and washed 4× with PBScontaining 0.1% Tween 20 (PBST). Reactions between immobilized substrateand mutant DhaAs were performed using purified GST-DhaA-Flag fusions.Various concentrations of protein (100 μL; diluted in PBSTB) wereincubated with immobilized substrate for various times at 25° C., andthe reactions were stopped by emptying the plates and washing 4× withPBST. To detect bound DhaA, 100 μL anti-GST-HRP (Amersham) was added toeach well at a 1:10,000 dilution (in PBSTB) and the plates incubated for1 hour at 25° C. Plates were emptied and washed 8× with PBST and thenTMB was added in a volume of 100 μL. After 15 minutes, color developmentwas stopped by the addition of an equal volume of 0.2 M H₂SO₄, andsignals were quantitated by measuring the absorbance at 450 nm.

Characterization of DhaA Mutants in Mammalian Cells.

Select sequence verified DhaA mutants were cloned into the mammalianexpression vector pCI-neo as follows: The DhaA-FLAG portion of themutant genes were removed from pGEX5X3 with SalI and NotI restrictionendonucleases. Fragments were separated by electrophoresis in 1% agarose(1×TAE), excised and purified using QIAquick Gel Extraction Kit(QIAGEN). The pCI-neo vector backbone was also digested with SalI andNotI, separated and purified in the same manner. Ligations wereperformed using Promega's LigaFast System, at an approximateinsert:vector ratio of 5:1. DNA was transformed into chemicallycompetent JM109 cells and plated onto LB agar plates containing Amp.Transformant colonies were picked into 96 well assay blocks (FisherScientific) containing 1 mL of LB+Amp and shaken overnight at 37° C.Cells were harvested and plasmids purified using the Wizard 96 plasmidpurification kit (Promega Corp.). Plasmids were screened for thepresence of the DhaA insert by a SalI-NotI restriction digest, andscreened by electrophoresis in 1% agarose (1×TAE). Positive clones wereverified by DNA sequence analysis.

Plasmid pHT2 was created to improve protein production in mammaliancells and to facilitate the generation of fusion proteins. DhaA.H272F YLwas amplified from pCIneo containing DhaA.H272F YL-FLAG witholigonucleotides 10055643 (5′CTA TAG GCT AGC CAG CTG GCG CGG ATA TCG CCACCA TGG GAT CCG AAA TCG GTA CAG GCT TCC CCT TCG 3′; SEQ ID NO:84) and10055644 (5′AGG GAA GCG GCC GCC TAC TTA ATT AAC TAT TAG CCG GCC AGC CCGGGG AGC CAG CGC GCG ATC TCA CTG C 3′; SEQ ID NO:85). The PCR product anddestination vector were both cut with EcoRV/NotI, gel purified, ligated,and transformed into JM109. The DhaA protein encoded by pHT2, designatedHT2, contained additional changes to the amino acid sequence ofDhaA.H272F YL. In addition to the H272F, K175M, C176G, and Y273Lsubstitutions, additional changes included: 1) a glycine insertion atposition 2 to generate a better Koazak sequence; 2) a Ala292Glysubstitution used to create a SmaI/XmaI/AvaI site; and an insertion ofalanine and glycine (AlaGly) to the C-terminus to generate a NaeI site(FIG. 49).

Mammalian Cell Culture.

CHO-K1 cells (ATCC-CCL61) or HeLa cells (ATCC-CCL2) were cultured in aHam's F12 nutrients or Dulbecco's modified minimal essential medium(respectably) supplemented with 10% fetal bovine serum (FBS), 100 U/mlpenicillin, and 100 mg/ml streptomycin, in an atmosphere of 95% air and5% CO₂ at 37° C.

Mammalian Cell Transfection.

To study transient expression of different proteins, cells were platedin 24 well plates (Labsystems) or 8 well LT cover glass chamber slides(Nunc) at a density of 30,000 cells/cm². At about 80-90% confluency, thecells were exposed to a mixture of lipofectamine/DNA/antibiotic freemedia according to the manufacturer's (Invitrogen) instructions. Thefollowing day, media was replaced with fresh media and cells wereallowed to grow for various periods of time.

Cell-to-Gel Analysis.

CHO-K1 cells were plated in 24 well plates (Labsystems) and transfectedwith a pCIneo-CMV.DhaA mutant-Flag vector. Twenty-four hours (in someexperiments 12, 24 or 48 hours) later, media was replaced with freshmedia containing 0.2, −25.0 μM carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Clor DiAc carboxyfluorescein-C₁₀H₂₁NO₂—Cl and the cells were placed into aCO₂ incubator for 1, 5, 15 or 60 minutes. Following this incubation,media was removed, cells were quickly washed with PBS (pH 7.4; twoconsecutive washes: 1.0 ml/cm²; 5 seconds each) and the cells weresolubilized in a sample buffer (1% SDS, 10% glycerol, and the like; 200μl/well). Proteins (2-10 μl/lane) were resolved on SDS-PAGE (4-20%gradient gels). Binding of the carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Clto proteins was quantified on a fluoroimager (Hitachi, Japan) atE_(ex)/E_(em) equal 540/575 nm.

Cell Imaging.

HeLa cells were plated in 8 well LT cover glass chamber slides (Nunc)and transfected with a pCIneo-CMV.DhaA mutant orβ-arrestin2-connector-DhaA.H11YL vector. Twenty-four hours later, mediawas replaced with fresh media containing different concentrations(0.2-10.0 μM) of carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl or DiAccarboxyfluorescein-C₁₀H₂₁NO₂—Cl and the cells were placed into a CO₂incubator for 15 minutes. Following this incubation, media was removedand cells were quickly washed with PBS (pH 7.4; two consecutive washes:1.0 ml/cm²; 5 seconds each). For live cells imaging experiments, newmedia was added to the cells. The cells were placed back into a CO₂incubator and after 60 minutes media was replaced with fresh media.Fluorescent images of the cells were taken on confocal microscopeFluorView500 (Olympus) with filter sets appropriate for the detection ofcarboxyfluorescein and carboxytetramethylrhodamine. To fix cells, 200 mlof 3.7% paraformaldehyde in PBS (pH 7.4) containing 0.1% Triton-X100 wasadded to the cells. After 15 minutes room temperature (RT), cells werewashed with PBS (pH 7.4) containing 1.0% Triton-X100 (10 minutes, RT).Detergent solution was replaced with PBS (pH 7.4), and images of thecells were taken on confocal microscope FluorView500 (Olympus). In someexperiments cells were counterstained with 100 nM of MitoTracker® GreenFM (Invitrogen, M-7514) or MitoTracker® Orange CMTMRos (Invitrogen,M-7510) for 15 minutes at 37° C.

Production of DhaA Fusions.

A β-arrestin2-connector-HT2 fusion cassette was constructed bysubcloning β-arrestin2 (See Example VII) into NheI/BamHI restrictionsites of the pHT2 vector (Promega). Two primers

(5′-CTATAGGCTAGCCAGCTGGCGCGGATATCGCCACCATGGGGGAGAAACCCGGGACCAGGG-3′; SEQ ID NO: 76, and5′-GATTTCGGATCCCATTCTAGAGGGCCCGCGGTACCGCAAGCTTGATCCGGAGCAGAGTTGATCATCATAGTCGTCATCC-3′; SEQ ID NO: 77)were designed to add a sequence encoding a connector and a BamHI site tothe 3′ end of β-arrestin2 coding region, and to amplify the fragmentfrom a β-arrestin2-connector-DhaA.H272F template.

The phRLuc-connector-HT2-Flag fusion cassette was constructed byreplacing the DhaA.H272F coding region in the vector encodingphRLuc-connector-DhaA.H272F-Flag (See Example IX) with the HT2 codingregion. Two primers

(5′-GCCCTCTAGAGCCGTCGACGCTGCCATGGGATCCGAAATCG-3′; SEQ ID NO: 78, and5′-GTAGTCACCGGTGCCGGCCAGCCCGGGGAGCCAGCGCGCG-3′; SEQ ID NO: 79)were designed to add a XbaI site to the 5′-end and a AgeI site to the3′-end of the coding region for HT2, and to amplify a 925 bp fragmentfrom a pHT2 template. The DhaA.H272F coding region was excised with XbaIand AgeI restriction enzymes, then the 925 bp fragment encoding HT2 wasinserted into the XbaI and AgeI sites of thephRLuc-connector-DhaA.H272F-Flag coding vector. The sequence of allfusion constructs was confirmed by DNA sequencing.

Renilla Luciferase-HT2-Flag Fusion Proteins Expressed in LivingMammalian Cells.

CHO-K1 cells were plated in 24 well plates (Labsystems) and transfectedwith a pCIneo.hRLuc-connector-HT2-Flag vector. Twenty-four hours later,media was replaced with fresh media containing 25 μM biotin-X-14-Cl and0.1% DMSO, or 0.1% DMSO alone, and the cells were placed in a CO₂incubator for 60 minutes. At the end of the incubation, the media wasremoved, cells were quickly washed with PBS (pH 7.4; two consecutivewashes; 1.0 ml/cm²; 5 seconds each) and new media was added to thecells. In some experiments, the media was not changed. The cells wereplaced back in a CO₂ incubator.

After 60 minutes, media was removed, and the cells were collected in PBS(pH=7.4, 200 ml/well, RT) containing protease inhibitors (Sigma #P8340).The cells were lysed by trituriation through a needle (IM1 23GTW). Then,cell lysates were incubated with Streptavidin Magnasphere ParamagneticParticles (Promega #Z5481) according to the manufacturer's protocol.Briefly, cell lysates were incubated with beads for 60 minutes at RTusing a rotating disk. Unbound material was collected; beads were washedwith PBS containing 0.5. % Triton-X100 (3×500 ml, pH=7.4, RT) andresuspended in SDS-sample buffer (for SDS-PAGE analysis) or PBS (pH=7.4,for determination of Renilla luciferase (R.Luc) activity). Proteins wereresolved on SDS-PAGE, transferred to a nitrocellulose membrane, analyzedwith anti-Flag-Ab, and bound antibody detected by an enhancedchemiluminescence (ECL) system (Pharmacia-Amersham). Activity of hR.Lucbound to beads was determined using Promega's “Renilla Luciferase AssaySystem” according to the manufacturer's protocol.

Results

Generating a Structural Model for DhaA.H272F

A structural model was built for DhaA.H272F using InsightII Modeler. Thereference structure for model calculation was 1BN6.pdb (Rhodococcusspecies DhaA). Five high-optimization models were calculated and onebest model selected based on the overall lowest energy and lowestviolations of structural parameters. The best model was thenstructurally aligned with the reference structure 1BN6.pdb to obtain ameasure of their overall and pair-wise differences, expressed as theRoot Mean Square Deviation (in Å) of aligned Cα atoms (FIG. 2A).

Identification of Substrate Tunnel

The structure of DhaA in the absence of substrate has been published andshows a buried active site cavity near the catalytic triad (FIG. 2A;Newman et al., 1999). However, it does not reveal the direction fromwhich the substrate enters the active site cavity (the “substratetunnel” or “ligand tunnel” herein). The likely location of the substratetunnel was identified by analyzing structures of related haloalkanedehalogenases complexed with different substrates (Protein Database). Inthese complexes, none of the substrates fill the entire ligand tunnel,but structural superimposition showed that the substrates were locatedat slightly different positions, which, taken together, allowedinference of the likely overall position of the substrate tunnel.Superimposition of the substrate-free DhaA structure (1BN6.pdb) thenallowed the identification of the corresponding substrate tunnelposition in DhaA.H272F.

Generation of DhaA-Substrate Model

A structural model of DhaA.H272F with a covalently attached substratewas generated (“DhaA-substrate model”). First,carboxyfluorescein-C₁₀H₂₁NO₂—Cl was manually docked into the substratetunnel of DhaA.H272F. Then a covalent bond was created between one ofthe carboxyl oxygens of the nucleophilic aspartate of DhaA and theterminal carbon of the substrate that becomes available after removal ofthe chloride (FIG. 2E). The length of this covalent bond was restrainedto about 3 Å to approximate the transition state. The covalentlyattached substrate was energy minimized separately and then togetherwith DhaA.H272F residues in the vicinity of the substrate. Energyminimizations were performed with Discover-3 using the CFF91 forcefield.

Identification of Residues for Mutagenesis

Residue numbering is based on the primary sequence of DhaA, whichdiffers from numbering in the published crystal structure (1BN6.pdb).Using the DhaA substrate model, dehalogenase residues within 3 Å and 5 Åof the bound substrate were identified. These residues represented thefirst potential targets for mutagenesis. From this list residues wereselected, which, when replaced, would likely remove steric hindrances orunfavorable interactions, or introduce favorable charge, polar, or otherinteractions. For instance, the Lys residue at position 175 is locatedon the surface of DhaA at the substrate tunnel entrance: removal of thislarge charged side chain might improve substrate entry into the tunnel(FIG. 2F). The Cys residue at position 176 lines the substrate tunneland its bulky side chain causes a constriction in the tunnel: removal ofthis side chain might open up the tunnel and improve substrate entry(FIG. 2F). The Val residue at position 245 lines the substrate tunneland is in close proximity to two oxygens of the bound substrate:replacement of this residue with threonine may add hydrogen bondingopportunities that might improve substrate binding (FIG. 2F). Lastly,Bosma et al. (2002) reported the isolation of a catalytically proficientmutant of DhaA with the amino acid substitution Tyr273Phe. Thismutation, when recombined with a Cys176Tyr substitution, resulted in anenzyme that was nearly eight times more efficient in dehalogenating1,2,3-trichloropropane (TCP) than the wild-type dehalogenase. Based onthese structural analyses, the codons at positions 175, 176 and 273 wererandomized, in addition to generating the site-directed V245T mutation.The resulting mutants were screened for improved rates of covalent bondformation with fluorescent (e.g., a compound of formula VI or VIII) andbiotin (FIG. 7) coupled DhaA substrates.

Library Generation and Screening

The starting material for all library and mutant constructions werepGEX5X3 based plasmids (FIG. 3A) containing genes encoding DhaA.H272Fand DhaA.D106C (FIG. 2B). These plasmids harbor genes that encode theparental DhaA mutants capable of forming stable covalent bonds withhaloalkane ligands. Codons at positions 175, 176 and 273 in theDhaA.H272F and DhaA.D106C templates were randomized using a NNKsite-saturation mutagenesis strategy. In addition to the single-sitelibraries at these positions, combination 175/176 NNK libraries werealso constructed. Sequence analysis of random clones from theselibraries, however, revealed the presence of a high (50%) frequency ofclones with unaltered wild-type sequences. Troubleshooting theQuikChange Multi protocol by varying template concentrations andextending the number and duration of the DpnI treatments did not have asignificant effect on this frequency. The rate of wild-type sequencecontamination in the libraries was, therefore, taken into account whendetermining the number of clones to screen from each library. Forexample, a single site NNK library has a codon diversity of 32 thatencodes all 20 amino acids. An approximately 5-fold oversampling of thelibrary is required to cover 99% of the possible sequence variants (L=−Vln 0.01). This oversampling translates into the need to screen at least160 individual clones. However, because the libraries were contaminatedto a significant extent by wild-type sequences (about 50%),approximately 400 clones from each single-site library were typicallyexamined. The combination 175/176 NNK NNK libraries had a theoreticalcodon diversity of 1024 encoding 400 different amino acid combinations.Approximately 3,000 to 4,000 clones from each double-site library wereexamined. In total, therefore, approximately 10,000 clones were selectedfor screening.

Three assays were evaluated as the primary screening tool for the DhaAmutant libraries. The first, an in vivo labeling assay, was based on theassumption that improved DhaA mutants in E. coli would have superiorlabeling properties. Following a brief labeling period withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl and cell wash, superior clonesshould have higher levels of fluorescent intensity at 575 nm. FIG. 31Ashows that screening of just one 96 well plate of the DhaA.H272F 175/176library was successful in identifying several potential improvements(i.e., hits). Four clones had intensity levels that were 2-fold higherthan the parental clone. Despite the potential usefulness of this assay,however, it was not chosen as the primary screen because of thedifficulties encountered with automation procedures and due to the factthat simple overexpression of active DhaA mutants could give rise tofalse positives.

The second assay that was considered as a primary screen was an in vitroassay that effectively normalized for protein concentration by capturingsaturating amounts of DhaA mutants on immobilized anti-FLAG antibody ina 96 well format. FIG. 31B shows the screening results obtained from oneplate of the DhaA.H272F 175/176 combination library using the proteincapture assay. Like the in vivo assay, this assay was also able toclearly identify potential improved DhaA mutants from a large backgroundof parental activities. Several clones produced signals up to 4-foldhigher than the parent DhaA.H272F. This assay, however, was costly dueto reagent expense and assay preparation time, and the automation ofmultiple incubation and washing steps. In addition, this assay wasunable to capture some mutants that were previously isolated andcharacterized as being superior.

The assay that was ultimately adopted as the DhaA primary screen wasbased on MagneGST™ protein purification resin (Promega Corp.). Anoverview of this in vitro screening assay is shown in FIG. 32. Briefly,cell pellets from cultures grown in 96 well plates were resuspended in areagent cocktail that contained lysis buffer, labeling reagent(substrate carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl) and MagneGST resin.This significantly streamlined the assay by combining lysis, labelingand protein capture in a single step. Following a brief incubationperiod with shaking the resin during which proteins were magneticallycaptured, the wells were washed prior to elution of the labeled DhaAmutants. The eluates were examined for fluorescence intensity at 580 nm.This streamlined screening assay was easily adapted onto an automatedTecan robotic platform that could examine about twenty 96 well plates ina 6.5 hour period.

The automated MagneGST™-based assay was used to screen the DhaA mutantprotein libraries. Screening of the DhaA.H272F and DhaA.D106C-based 175single-site libraries failed to reveal hits that were significantlybetter than the parental clones (data not shown). FIGS. 33A-B showrepresentative screening results of the 176 single-site and 175/176combination libraries, respectively. The screen identified severalclones with superior labeling properties compared to the parentalcontrols. FIG. 34 shows two representative screening plates from theDhaA.H272F Y273 NNK library. Three clones with significantly higherlabeling properties could be clearly distinguished from the backgroundwhich included the DhaA.H272F parent. For clones with at least 50%higher activity than the DhaA.H272F parent, the overall hit rate of thelibraries examined varied from between 1-3%. Similar screening resultswere obtained for the DhaA.D106C libraries (data not shown). The hitsidentified by the initial primary screen were located in the masterplates, consolidated, re-grown and reanalyzed using the MagneGST™ assay.Only those DhaA mutants with at least a 2-fold higher signal than theparental control upon reanalysis were chosen for sequence analysis.

Sequence Analysis of DhaA Hits

FIG. 35A shows the codons of the DhaA mutants identified followingscreening of the DhaA.H272F libraries. This analysis identified sevensingle 176 amino acid substitutions (C176G, C176N, C176S, C176D, C176Tand C176A, and C176R). Interestingly, three different serine codons wereisolated. Numerous double amino acid substitutions at positions 175 and176 were also identified (K175E/C176S, K175C/C176G, K175M/C176G,K175L/C176G, K175S/C176G, K175V/C176N, K175A/C176S, and K175M/C176N).While seven different amino acids were found at the 175 position inthese double mutants, only three different amino acids (Ser, Gly andAsn) were identified at position 176. A single K175M mutation identifiedduring library quality assessment was included in the analysis. Inaddition, several superior single Y273 substitutions (Y273C, Y273M,Y273L) were also identified.

FIG. 35B shows the mutated codons of the DhaA mutants identified in theDhaA.D106C libraries. Except for the single C176G mutation, most of theclones identified contained double 175/176 mutations. A total of 11different amino acids were identified at the 175 position. In contrast,only three amino acids (Gly, Ala and Gln) were identified at position176 with Gly appearing in almost ¾ of the D106C double mutants.

Characterization of DhaA Mutants

Several DhaA.H272F and D106C-based mutants identified by the screeningprocedure produced significantly higher signals in the MagneGST assaythan the parental clones. FIG. 36A shows that the DhaA.H272F basedmutants A7 and H11, as well as the DhaA.D106C based mutant D9, generatea considerably higher signal withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl than the respective parents. Inaddition, all of the DhaA.H272F based mutants identified at the 273position (Y273L “YL”, Y273M “YM”, and Y273C “YC”) appeared to besignificantly improved over the parental clones (FIG. 36B) using thebiotin-PEG4-14-Cl substrate. The results of these analyses wereconsistent with protein labeling studies using SDS-PAGE fluorimage gelanalysis (data not shown). In an effort to determine if combinations ofthe best mutations identified in the DhaA.H272F background wereadditive, the three mutations at residue 273 were recombined with theDhaA.H272F A7 and DhaA.H272F H11 mutations. In order to distinguishthese recombined protein mutants from the mutants identified in roundone of screening (first generation), they are referred to as “secondgeneration” DhaA mutants.

To facilitate comparative kinetic studies several improved DhaA mutantswere selected for purification using a Glutathione Sepharose 4B resin.In general, production of DhaA.H272F and DhaA.D106C based fusions in E.coli was robust, although single amino acid changes may have negativeconsequences on the production of DhaA (data not shown). As a result ofthis variability in protein production, the overall yield of the DhaAmutants also varied considerably (1-15 mg/mL). Preliminary kineticlabeling studies were performed using several DhaA.H272F derivedmutants. FIG. 37A shows that many, if not all, of the mutants chosen foranalysis had faster labeling kinetics than the H272F parent. In fact,upon closer inspection of the time course, the labeling of several DhaAmutants including the first generation mutant YL (lane 15) and the twosecond generation mutants, A7YM and H11YL (lanes 13 and 21,respectively) mutants appeared to be complete by 2 minutes. A moreexpanded time course analysis was performed on the DhaA.H272F A7 and thetwo second generation DhaA.H272F mutants A7YM and H11YL. As is evidentfrom FIG. 37B, the labeling reactions of the two second generationclones are for the most part complete by the first time point (20seconds). The A7 mutant, on the other hand, appears only to be reachingcompletion by the last time point (7 minutes). The fluorescent bands ongel were quantitated and the relative rates of product formation areshown in FIG. 37C. In order to determine a labeling rate, theconcentration of the H11YL was reduced from 50 ng to 10 ng and a morerefined time-course was performed. The results shown in FIG. 38Ademonstrate that under these labeling conditions a linear initial ratecan be measured. Quantitation of the fluorimaged gel data allowed secondorder rate constants to be calculated (FIG. 38B). Based on the slopeobserved, the second order rate constant forcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl labeling of DhaA.H272F H11YLwas 5.0×10⁵ M⁻¹ sec⁻¹.

Fluorescence polarization (FP) is ideal for the study of smallfluorescent ligands binding to proteins. It is unique among methods usedto analyze molecular binding because it gives direct nearlyinstantaneous measure of a substrate bound/free ratio. Therefore, an FPassay was developed as an alternative approach to fluorimage gelanalysis of the purified DhaA mutants. FIG. 39A shows the relativelabeling rate of the H272F parent, compared to the A7 and H11YL mutants.Under the labeling conditions used in this experiment, the secondgeneration mutant DhaA.H272F H11YL was significantly faster than its A7and H272F counterparts. To place this rate in perspective, approximately42 and 420-fold more A7 and parental, i.e., DhaA.H272F, protein,respectively, was required in the reaction to obtain measurable rates.FIG. 39B shows the FP results using carboxyfluorescein-C₁₀H₂₁NO₂—Cl.Under the labeling conditions used in this experiment, it is evidentthat the H11YL mutant was also considerably faster than A7 and parental,DhaA.H272F proteins with the fluorescein-based substrate. However, itappears that labeling of H11YL with carboxyfluorescein-C₁₀H₂₁NO₂—Cl ismarkedly slower than labeling with the correspondingcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl substrate. Four-fold more H11YLprotein was used in the carboxyfluorescein-C₁₀H₂₁NO₂—Cl reaction (150nM) versus the carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl reaction (35nM), yet the rate observed in FIG. 39B appears to be qualitativelyslower than the observed carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl rateshown in FIG. 39A.

Based on the sensitivity and truly homogenous nature of this assay, FPwas used to characterize the labeling properties of the purified DhaAmutants with the fluorescently coupled substrates. The data from thesestudies was then used to calculate a second order rate constant for eachDhaA mutant-substrate pair. The results of these analyses are shown inFIG. 40. The two parental proteins used in this study, DhaA.H272F andDhaA.D106C, were found to have comparable rates with thecarboxytetramethylrhodamine and carboxyfluorescein-based substrates.However, in each case labeling was slower with thecarboxyfluorescein-C₁₀H₂₁NO₂—Cl substrate. All of the first generationDhaA mutants characterized by FP had rates that ranged from 7 to3555-fold faster than the corresponding parental protein. By far, thebiggest impact on labeling rate by a single amino acid substitutionoccurred with the three replacements at the 273 position (Y273L, Y273M,and Y273C) in the DhaA.H272F background. Nevertheless, in each of thefirst generation DhaA.H272F mutants tested, labeling with thecarboxyfluorescein-C₁₀H₂₁NO₂—Cl substrate always occurred at a slowerrate (1.6 to 46-fold). Most of the second generation DhaA.H272F mutantswere significantly faster than even the most improved first generationmutants. One mutant in particular, H11YL, had a calculated second orderrate constant with carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl that wasover four orders of magnitude higher than the DhaA.H272F parent. TheH11YL rate constant of 2.2×10⁶ M⁻¹ sec⁻¹ was nearly identical to therate constant calculated for a carboxytetramethylrhodamine-coupledbiotinistreptavidin interaction (FIG. 41). This value is consistent withan on-rate of 5×10⁶ M⁻¹ sec⁻¹ determined for a biotin-streptavidininteraction using surface plasmon resonance analysis (Qureshi et al.,2001). Several of the second generation mutants also had improved rateswith the carboxyfluorescein-C₁₀H₂₁NO₂—Cl substrate, however, as notedpreviously, these rates were always slower than with thecarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl substrate. For example, thecarboxyfluorescein-C₁₀H₂₁NO₂—Cl labeling rate of the DhaA.H272F H11YLmutant was 100-fold lower than thecarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl labeling rate.

Structure Analysis of the Improved DhaA.H272F H11YL Mutant.

Structural models were built for DhaA.H272F and DhaA.H272F H11YL usingInsightII Modeler. The reference structure for model calculation was1BN6.pdb (Rhodococcus species DhaA). Reference structures of twoadditional related haloalkane dehalogenases were included forcalculation of the DhaA.H272F H11YL model: 1CV2.pdb (Sphingomonaspaucimobilis) and 2DHD.pdb (Xanthobacter autotrophicus). For eachsequence, five high-optimization models were calculated and one bestmodel selected based on the overall lowest energy and lowest violationsof structural parameters. These best models were then structurallyaligned with the reference structure 1BN6.pdb to obtain a measure oftheir overall and pair-wise differences, expressed as the Root MeanSquare Deviation (in Å) of aligned Cα atoms.

The substrate carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl was covalentlyattached to the best structural models of DhaA.H272F and DhaA.H272FH11YL. First, the substrate was manually docked into the substratetunnel, and then a covalent bond was created between one of the carboxyloxygens of the nucleophilic aspartate of the protein and the terminalcarbon of the substrate that becomes available after removal of thechloride. Substrate conformations were adjusted to be as similar aspossible for both models. The initial models of DhaA.H272F andDhaA.H272F H11YL without and with covalently attached substrate werethen prepared for energy minimization by adding hydrogens at pH 7.0 andassigning potentials using the CFF91 force field. Both models wereenergy minimized with Discover-3 using non-bond interactions withgroup-based or atom-based cutoffs, a distance-dependent dielectric of1.0, and a final convergence of 0.01 for the last minimization step. Thefollowing minimization cascade was used for models without substrate: a)minimize hydrogens of whole system and fix other atoms, b) minimize sidechains of residues within about 8 Å of substrate and fix other atoms, d)minimize residues within about 8 Å of substrate with harmonic Cαrestraint and fix other atoms. This minimization cascade was used formodels with substrate: a) minimize hydrogens of whole system and fixother atoms, b) minimize substrate and fix other atoms, c) minimizesubstrate plus side chains of residues within about 8 Å of substrate andfix other atoms, d) minimize substrate plus residues within about 8 Å ofsubstrate with harmonic Cα restraint and fix other atoms. For allminimized models, bump checks were performed between the substrate andresidues within about 8 Å of substrate to determine steric hindrances.The substrate tunnel shape and size was visualized by calculating aConnolly surface with default probe radius of 1.4 Å for residues withinabout 5 Å of the substrate. All models were superimposed structurally toevaluate changes in the position of specific residues.

Position of Relevant Residues.

The nucleophile Asp106 moves slightly more into the tunnel upon bindingof carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl in both mutants. W107,located next to nucleophile and responsible for holding substrate boundto active site in proper orientation for nucleophilic attack, does notchange its position significantly. In DhaA.H272F, the F272 side chain issticking into the tunnel in the absence of substrate, and rotates out ofthe tunnel about 45° in the presence of substrate. In DhaA.H272F H11YL,the F272 side chain does not stick into the tunnel and adjusts itsposition only slightly in the presence of substrate. This shouldfacilitate substrate binding in DhaA.H272F H11YL. Glu130 shows a similarorientation in all structures except for DhaA.H272F with substrate,where the Glu130 side chain is pushed away from the tunnel by the F272side chain rotation necessary to accommodate the substrate.

Overall Fit of Substrate into Substrate Tunnel.

A bump check was performed of the minimized protein-substrate structuresto show which atoms of the substrate “bump” into which atoms of theprotein. A bump exists when two atoms overlap with at least 10% of theirvan der Waals radii. Carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl showsbumps to Lys175 and Cys176 in DhaA.H272F, but no bumps to any residuesin DhaA.H272 H11YL. This suggests that the mutations introduced inDhaA.H272F H11YL have widened the tunnel to some degree.

Substrate Tunnel Shape and Size.

The substrate cavity was visualized as a Connolly surface with defaultprobe radius of 1.4 Å. In the absence of substrate, DhaA.H272F shows adistinct tunnel entrance and a large cavity near the catalytic triad,separated by a strong constriction or discontinuity in the tunnel aroundCys176 extending to Tyr273 (FIG. 42A). This constriction is pushed openwhen the substrate is bound (FIG. 42B). Mutant DhaA.H272F H11YL does notshow any tunnel constriction at positions 176 and 273 but has acontinuously wide and open tunnel both in the absence (FIG. 42C) andpresence (FIG. 42D) of substrate, suggesting very easy substrate entry.The K175M mutation in DhaA.H272F H11YL does not seem to contributesignificantly to the opening of the tunnel.

Thermostability Studies with DhaA Mutants.

The thermostability profiles of selected first and second generationDhaA.H272F mutants were determined by measuring the residual activity ofthe purified proteins following 60 minute incubations at varioustemperature. FIG. 43A shows the thermostability profiles of the firstgeneration DhaA.H272F mutants and corresponding parent. The most activefirst generation mutants (DhaA.H272F YL, DhaA.H272F YC and DhaA.H272FYM) were relatively unstable at temperatures above 30° C. This is incontrast to the DhaA.H272F parent and the DhaA.H272F A7 mutant proteinthat were stable up to temperatures of 40° C. One mutant, DhaA.H272FH11, retained significant labeling activity following incubation as highas 50° C. (half-life of 58 minutes at 50° C.). Of the second generationDhaA.H272F mutants, DhaA.H272F H11YL retained the most activityfollowing incubation at 42° C. (FIG. 43B), however, certainly not to thedegree of DhaA.H272F H11 (FIG. 43A). It is likely that the samemutations that confer thermostability on DhaA.H272F H11 (i.e., K175M andC176G) also contribute to the stabilization of the DhaA.H272FYL mutant.

Effect of Temperature on DhaA.H272F H11YL Reaction Kinetics.

To examine the effect of temperature on reaction rates a labeling timecourse experiment was performed at room temperature and on ice (0° C.).Fluorimage gel anlaysis shows that the lower temperature does not impairthe labeling rate with carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl (FIG.44). In fact, the rate at the lower reaction temperature appears toproceed at a faster rate. Calculation of the 2^(nd) order rate constantfor the 0° C. reaction reveals a rate of 3.1×10⁶ M⁻¹ sec⁻¹ compared to5×10⁵ M⁻¹ sec⁻¹ for the reaction incubated at 25° C.

Reaction Between DhaA Mutants and Immobilized Biotin ChloroalkaneSubstrates.

In order to investigate how well the improved DhaA mutants react with animmobilized substrate, an ELISA-type assay utilizing pre-coatedstreptavidin plates was employed (FIG. 45A). Eight DhaA.H272F mutants(A7, H11, YL, YM, H11YL, H11YM, A7YL and A7YM) were titrated againstthree different biotin containing substrates (FIG. 7). Thebiotin-PEG4-14-Cl results shown in FIG. 45B indicate that bothDhaA.H272F YL and DhaA.H272F YM mutant proteins react most efficientlywith that substrate. In addition, both DhaA.H272F A7YL and DhaA.H272FA7YM were more efficient than DhaA.H272F H11YL and DhaA.H272F H11YM.None of the best performing clones with the biotin-PEG14-14-Cl substratebound as well to the other two biotin substrates, suggesting thatbiotin-PEG4-14-Cl is a preferred substrate (data not shown). The firstgeneration DhaA mutants, DhaA.H272F A7 and H11, reacted poorly with allbiotin substrates tested.

Characterization of DhaA Mutants in Mammalian Cells.

In Vivo and In Vitro Labeling of DhaA Mutants.

The production of some DhaA mutant proteins in E. coli was compromisedat 37° C., while other improved DhaA mutants retained considerableactivity when grown and induced at elevated temperatures. These clonesmay have a selective folding advantage at higher temperatures, and, as aresult, may therefore be able to better tolerate mammalian cell cultureconditions. Based on their superior kinetic and/or productionperformance, genes encoding the mutant proteins DhaA.H272F A7 and H11(along with the two parents DhaA.H272F and DhaA.D106C) were cloned intothe mammalian expression vector pCI-neo and transfected into CHO cells.A kinetic, in vivo labeling study showed that the two first generationmutants DhaA.H272F A7 and H11, demonstrated superior performancecharacteristics compared to parent DhaA.H272F at substrateconcentrations of 5 μM (FIGS. 46A and B). Therefore, the ability of theDhaA.H272F mutants A7 and H11 to retain significant activity/productionat 37° C. in E. coli correlated well with its superior performance inmammalian cells.

Three additional DhaA mutants were tested forcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl labeling efficiency intransiently expressing CHO-K1 cells. FIGS. 46C-D show the labelingresults comparing DhaA.H272F A7, DhaA.H272F H11YL and DhaA.D106C 30H4.At a carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl substrate concentration of5 μM, the second generation DhaA.H272F H11YL was labeled to completionin 15 minutes. This was half the time it required for complete labelingof DhaA.H272F A7. By contrast, DhaA.D106C 30H4 (the DhaA.H272F H11equivalent in the DhaA.D106C background) required over 2 hours toachieve the same degree of labeling.

The dependence of labeling efficiency on substrate concentration withDhaA.H272F A7 and H11YL was also investigated. FIGS. 46A-C demonstratethe superior labeling properties of DhaA.H11YL in mammalian celllysates, particularly at low carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Clsubstrate concentrations (i.e., 0.1 and 1.0 μM). This finding isconsistent with the results of in vitro kinetic studies using purifiedDhaA proteins. Slightly slower binding kinetics of DhaA.H272F H11YL tocarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl were observed in vivosuggesting that the mammalian cell membrane may be limiting transport ofthe fluorescent ligand into cells (data not shown).

In Vivo Stability of DhaA Mutants.

The stability of select DhaA mutants in transiently transfectedmammalian cells was investigated. FIG. 48A shows the fluorescent signalobtained from the parental and two first generation mutants DhaA.H272FA7 and DhaA.H272F H11, after labeling cells withcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl 12, 24, and 48 hourspost-transfection. Quantitation of the fluorimage gel shows that theproduction of active protein from all four clones tested peaks at 24hours post-transfection and then declined to the levels observed at 48hours (FIG. 48B). However, CHO-K1 cells transfected with constructsencoding the H272F-derived mutants A7 and H11 retained the ability toproduce more active protein after 48 hours than either of the twoparental mutants. This is clearly evident from the robust fluorescentsignal produced after carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl labeling.This result suggests that the DhaA.H272F A7 and H11 mutants may besignificantly more stable in vivo. FIGS. 47C-D show a similar stabilityanalysis comparing DhaA.H272F A7 with the second generation mutantDhaA.H272F H11YL. CHO-K1 cells transfected with the construct encodingthe DhaA.H272F H11YL mutant also retained a significant labelingpotential at 48 hours. In fact, there was little to no detectablereduction in the signal produced by DhaA.H272F H11YL during the 24-48hour period.

Imaging of DhaA.H272F H11YL in Live and Fixed Mammalian Cells.

As shown by the images in FIGS. 50A-B, DhaA.H272F H11YL expressed inmammalian cells could be efficiently labeled bycarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl orDiAc-carboxyfluorescein-C₁₀H₂₁NO₂—Cl. Images are bright and showexcellent signal-to-noise ratio. As shown by the images in FIGS. 50C-D,DhaA.H272F H11YL HT2 (FIG. 49)and DhaA.H272F could be efficientlylabeled with TAMRA-C₁₁H₂₁N₁O₃—Cl, and fixed with 3.7% paraformaldehyde.Images of the cells expressing DhaA.H11YL HT2 and stained with 0.2, 1.0or 5.0 μM TAMRA-C₁₁H₂₁N₁O₃—Cl for 5 minutes are brighter than images ofthe cells expressing DhaA.H272F and stained with 5.0 μMcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl for 30 minutes. This stronglyindicates that in mammalian cells,carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl labels DhaA.H272F H11YL HT2with higher efficiency than DhaA.H272F.

Imaging of β-Arrestin2-Connector-DhaA.H272F H11YL HT2 Fusion ProteinExpressed in Living Mammalian Cells.

As shown by the images in FIGS. 50E-F, β-arrestin2-connector-DhaA.H272FH11YL HT2 expressing cells have a typical cytosolic localization forβ-arrestin2 using either DiAc-carboxyfluorescein-C₁₀H₂₁NO₂—Cl orcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl to label the protein fusion.

Capturing of DhaA.H272F H11YL-Renilla Luciferase Fusion ProteinExpressed in Living Mammalian Cells.

As shown in FIGS. 51A-B, significant luciferase activity was detected onbeads incubated with a lysate of cells treated with biotin-X-14-Cl andexcess of biotin-X-14-Cl was washed out. No luciferase activity wasdetected on beads incubated with a lysate from cells that were nottreated with biotin-X-14-Cl. Moreover, no hR.Luc activity was detectedon beads incubated with lysate from the cells treated withbiotin-X-14-Cl when free biotin-X-14-Cl was not washed out. Takentogether, these data show that functionally active protein (hR.Luc)fused to the DhaA.H272F H11YL HT2 can be efficiently captured usingbiotin-X-14-Cl and SA-coated beads. The capture is biotin-dependent, andcan be competed-off by excess of biotin-X-14-Cl. As a significantinhibitory effect of the beads on the hR.Luc activity was observed (datanot shown), SDS-PAGE and Western blot analysis with anti-R.Luc antibodywere used to estimate the efficiency of capture ofhR.Luc-connector-DhaA.H272F H11YL HT2 fusion protein. As shown in FIG.51B, more than 50% of hR.Luc-connector-DhaA.H272F H11YL HT2 fusionprotein can be captured in a biotin-dependent manner.

Reactivity of DhaA.H272F H11YL with Haloalkane Substrates ContainingModified Linkers.

The substrate cavity of the Rhodococcus dehalogenase (DhaA) protein issignificantly larger, in both length and breath, than the substratetunnel of the Xanthobacter DhlA protein (Newman et al., 1999). As aresult the labeling technology, DhaA mutants should be capable ofaccommodating a range of substrates containing different linkerstructures. Some examples of alternative substrates include thep-phenethyl and furanyl propyl derivatives, e.g., a compound such asthose shown in FIG. 56. The reactivity of these modified haloalkanesubstrates was tested with the purified DhaA.H272F H11YL protein.

FIG. 52A shows the binding rates of variouscarboxytetramethylrhodamine-based substrates determined using FPanalysis. The apparent binding rate constant determined for interactionof the carboxytetramethylrhodamine-p-phenethyl-Cl substrate toDhaA.H272F H11YL was only 3-fold lower than the rate determined forcarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl. However, no binding wasdetected for the carboxytetramethylrhodamine-furanyl propyl substrateunder these reaction conditions. The relative labeling rates of thecarboxytetramethylrhodamine-based substrates was confirmed usingfluorimage gel analysis. Under the reaction conditions used, all threecarboxytetramethylrhodamine substrates were found to react with theprotein (FIG. 52B). The fluorescent bands on the gel were quantitated todetermine the relative rates of product formation. A comparison of theslopes of product accumulation shows that thecarboxytetramethylrhodamine-p-phenethyl-Cl substrate was significantlyslower at labeling DhaA.H272F H11YL than thecarboxytetramethylrhodamine-C₁₀H₂₁NO₂—Cl substrate (FIG. 52C). Thecarboxytetramethylrhodamine-furanyl-propyl-Cl substrate was over100-fold slower than the carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Clsubstrate.

A similar in vitro labeling experiment was performed usingcarboxyfluorescein modified p-phenethyl and furanyl substrates. FIG. 53shows the relative binding rates of the various carboxyfluorescein-basedsubstrates using FP analysis. The apparent binding rate constantdetermined for the carboxyfluorescein-p-phenethyl-Cl substrate (5.6×10³M⁻¹ sec⁻¹) was approximately 5-fold lower than that forcarboxyfluorescein-14-Cl (FIG. 53A). As previously observed with thecarboxytetramethylrhodamine chloroalkane binding experiments, no bindingwas detected for the carboxyfluorescein-furanyl substrate under thesereaction conditions. The relative labeling rates of thecarboxyfluorescein-based substrates was also determined using fluorimagegel analysis. FIG. 53B shows the amount of fluorescent product formedover the course of 20 minutes. Under the reaction conditions used allthree carboxyfluorescein substrates were found to react with the protein(FIG. 53B). Quantitation of these product bands revealed that theDhaA.H272F H11YL labeled approximately 3-fold slower with thecarboxyfluorescein-p-phenethyl-Cl substrate compared to thecarboxyfluorescein-C₁₀H₂₁NO₂—Cl substrate (FIG. 53C). However, thelabeling rate with the carboxyfluorescein-furanyl-propyl-Cl substratewas over 100-fold slower than the carboxyfluorescein-C₁₀H₂₁NO₂—Clsubstrate.

The in vivo labeling rates of the variouscarboxytetramethylrhodamine-based substrates was determined in mammaliancells. CHO-K1 cells transiently transfected with pHT2 vector (DhaA.H272FH11YL) were labeled with different concentrations ofcarboxytetramethylrhodamine-Cl-alkanes for over a time course of 60minutes. Cells were collected at various times, lysed, and proteins wereresolved on SDS-PAGE. The presence of labeled protein was detected witha fluoroimager. FIG. 54A shows the accumulation of labeled product overtime at various substrate concentrations of 1, 5 and 20 μM. Quantitationof fluorescent product accumulation demonstrates that labeling ofDhaA.H272F H11YL with carboxytetramethylrhodamine-p-phenethyl-Clsubstrate was comparable to the carboxytetramethylrhodamine-C₁₀H₂₁NO₂—Clsubstrate at all concentrations tested (FIG. 54B). The labeling rate ofthe DhaA.H272F H11YL mutant with thecarboxytetramethylrhodamine-furanyl-propyl-Cl substrate, however, wasnoticeably slower at the 1 and 5 μM substrate concentrations.

The biotin-p-phenethyl-Cl substrate was tested in its ability to reactwith immobilized DhaA protein. The general reaction scheme for the ELISAtype assay performed is shown in FIG. 55A. Two pmol of DhaA.H272F H11YLwas immobilized onto wells of a microtiter plate using anti-FLAGantibody. Following incubation with the haloalkane substrates (17 μM)and washing, the bound substrate was detected using a streptavidin-HRPconjugate. The amount of color after development was an indication ofthe reactivity of each biotin haloalkane substrate. FIG. 55B shows thatthe biotin-p-phenethyl substrate reacted with the immobilized DhaAprotein but to a lesser extent than either the biotin-14-Cl andbiotin-PEG4-14-Cl substrates.

Example XI Exemplary DhaA Fusions for Cell Surface Display

Many membranous enzymes, receptors, differentiation antigens and otherbiologically active proteins are bound to fatty acids, isoprenoids,diacylglycerols, and glycosylphosphatidylinositols (GPI) throughpost-translational processing, and anchored to the membrane by theselipids. GPI-linked proteins are expressed on a wide variety of celltypes and have diverse functions ranging from control of cell adhesion(e.g., CD48, CD58, Thy-1/CD90) to protection against complement (CD55,CD59) and enzyme activity (alkaline phosphotase). These molecules areunique in that they are anchored to the outer leaflet of the plasmamembrane only and thus do not extend into the cytoplasm. Withoutexception, GPI anchors are covalently linked to carboxyl-terminal endsof proteins. The core structure for GPI anchors in eukaryotes iscomposed of ethanolamine phosphate, trimannoside, glucosamine andinositol phospholipid in that order. All known GPI-anchored proteins aresynthesized with a C-terminal cleavable peptide (reviewed in Stevens,1995; Tiede et al., 1999; Sevelever et al., 2000). The C-terminalpeptide (a) is comprised of 15-30 amino acids that are generallyhydrophobic, (b) contains no downstream cytosolic domain (Medof andTykocinski, (1990), and (c) establishes a pattern defined by certainsets of amino acids around the “cleavage-attachment” site. This site,which is the amino acid left after removal of the C-terminal signal andthe attachment of the GPI anchor, has been termed the ω amino acid.

GPI is synthesized by sequential addition of sugars and ethanolaminephosphates to phosphatidylinositol in the endoplasmic reticulum (ER)(Udenfriend and Kodukula, 1995; Kinoshita and Inoue, 2000). The backbonestructure of GPI is common among different species. Pre-formed GPI isattached to proteins in the ER. Precursor proteins to be modified withGPI have two signals. One at the N-terminus is a signal required fortranslocation across the ER membrane. The other, at the C-terminus, is aGPI attachment signal. The GPI attachment signal peptide is recognizedby the GPI transamidase, which cleaves the signal peptide and replacesit with GPI.

To generate a GPI-anchored DhaA mutant, a strategy suggested by DeAngelis et al. (1998) for generation of GPI-anchored GFP may beemployed. This strategy requires an additional N-terminal leader peptidefor directing the nascent polypeptide through to the ER membrane, andaddition of a C-terminal sequence for GPI attachment, e.g.,PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO:18). Using thisstrategy, Hiscox et al. (2002) successfully expressed GFP on the surfaceof CHO cells. The authors used three-stage PCR to ligate GFP downstreamof the signal peptide of human CD59

(amino acids −25 to 1, e.g., MGIQGGSVLFGLLLVLAVFCHSGHSL; SEQ ID NO: 25)and upstream of amino acids 67-102 of human CD59,e.g., FEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLEN (SEQ ID  NO: 44),which contains the GPI attachment site at residue 77.

GFP and DhaA have a drastically different structure. Therefore, togenerate GPI-anchored DhaA mutant fusions for mammalian cells, a signalsequence and GPI attachment sequence of different GPI-anchored proteins,e.g. 5′-nucleotidase (CD73), CAMPATH (CD52), the decay acceleratingfactor (DAF or DC55), the membrane inhibitor of reactive lysis (CD59),leucocyte function associated protein-3 (LFA-3 or CD90), placentalalkaline phosphatase (PLAP), acethylcholinesterase (AchE), Thy-1 (CD90),Prion, and the like, may be employed. To improve accessibility ofsubstrates to the catalytic pocket, a peptide connector may beintroduced between DhaA and the GPI attachment sequence.

Integrins are the major receptors connecting cells to the surroundingextracellular matrix (Danen & Yamada, 2001; Hohesnester & Engel, 2002).They not only support cell attachment but also act in concert withreceptors for soluble factors to regulate survival, differentiation, andproliferation. In vitro, integrin α5β1-mediated cell adhesion tofibronectin is particularly efficient in supporting mitogen-dependentproliferation of fibroblastic, epithelial, and endothelial cells.Integrins are heterodimeric transmembrane receptors connected viascaffolding proteins to the cortical actin cytoskeleton. Theextracellular regions of the α and β subunits are non-covalently linkedto form a globular head domain that binds specific extracellular matrix(ECM) with specificity determined by the particular combination of α andβ subunits. Sequencing of the human genome has identified as many as 24αand 9β subunits, and 24 different functional integrins are currentlyknown to exist in mammals.

To express DhaA mutants on the cell surface, a fusion of DhaA mutant andan integrin, e.g., an α or β integrin, is employed. Such a fusionprotein includes a transmembrane domain, cytosolic domain, and/or anextracellular stalk domain of integrin, and a DhaA mutant. The cytosolicdomain of integrin may be a truncated domain, and an extracellular stalkdomain of integrin may be replaced with an extracellular stalk domain ofanother protein (e.g., fractalkine), a portion of a stalk domain and/ora genetically engineered peptide, e.g., a synthetic peptide. Fusions ofintegrins with other proteins of interest, e.g., reporter proteins suchas GFP, or enzymes such as luciferase, is also envisioned, e.g., forcell surface display of the protein of interest.

The cadherins comprise a family of calcium-dependent cell adhesionmolecules that form and maintain adhesive contacts between cells ofsolid tissues (Takeichi et al., 1981; Hatta and Takeichi, 1986; Hatta etal., 1998). Cadherins are single-pass transmembrane proteinscharacterized by the presence of distinctive cadherin repeat sequencesin their extracellular segment (Patel et al., 2003). Each of theserepeats, consisting of 110 amino acids, forms a beta-sandwich domain.Cadherins typically have several of these “cadherin domains” tandemlyrepeated in their extracellular segments. The connections between thesedomains are rigidified by the specific binding of three Ca²⁺ ionsbetween each successive domain pair. Cadherins can be classified intoseveral subfamilies (Nollet et al., 2000): type I (classical) and typeII cadherins, which are ultimately linked to the actin cytoskeleton; thedesmosomal cadherins (desmocollins and desmogleins), which are linked tointermediate filaments; and the protocadherins, which are expressedprimarily in the nervous system. In addition, several “atypical”cadherins, proteins containing one or more cadherin repeat sequences butbearing no other hallmarks of cadherins, have also been described.

To express DhaA mutants on the cell surface, a fusion of DhaA mutant anda cadherin, e.g., cadherin type I, cadherin type II, or atypicalcadherin, is employed. Such a fusion protein includes a transmembranedomain, cytosolic domain, one or more extracellular cadherin domains,and a DhaA mutant. The cytosolic domain of cadherin may be a truncateddomain, and an extracellular cadherin domain(s) may be removed orreplaced with an extracellular stalk domain of another protein orgenetically engineered peptide. Truncated cadherin, T-cadherin, is atype of cadherin and is unusual because it lacks a transmembrane segmentand the conserved W2, but has a GPI anchor.

To express DhaA or a DhaA fusion on a cell surface, an N-terminal leaderpeptide for directing the nascent polypeptide through the phospholipidbilayer of membrane (e.g., ER membrane) is needed. The N-terminal leaderpeptide may be a leader peptide of the fusion partner of a DhaA fusionpolypeptide or a leader peptide of another polypeptide. In oneembodiment, an additional peptide e.g., a connector) may be insertedbetween DhaA and the N-terminal leader peptide, DhaA and thetransmembrane domain of a fusion partner, and/or DhaA and anextracellular domain(s) of a fusion partner.

Generally, to express DhaA mutants on the cell surface, a fusion of aDhaA mutant and any membrane protein that has a defined N-terminalextracellular domain(s) (e.g., ligand-gated ion channels such asn-methyl-D-aspartate (NMDA) receptors; 5-methyl-4-isoxazolopropionicacid (AMPA) receptors, glycine receptors, nicotinic acetylcholinereceptors (nAChRs), P2X receptors, 5-hydroxytryptamine-3 (5-HT3)receptors) (for review see Galligan, 2002), may be employed. In oneembodiment, a fusion of a DhaA mutant and any membrane protein that hasan extracellular C-terminal domain (e.g., inhibitory glycine receptors,for a review see Breitinger and Becker, 2002) is employed. The DhaA isattached to or inserted into C-terminal domain of the protein. Toimprove performance of the fusion (e.g., accessibility of Cl-alkaneligands to the catalytic pocket of DhaA), a peptide connector might beintroduced between DhaA and the C-terminal domain of protein. In anotherembodiment, a fusion of DhaA mutant and any membrane protein that has anextracellular loop domain (peptide chains connecting transmembranedomains of the protein, e.g., peptide chains connecting S1 and S2, S3and S4, S5 and S6 transmembrane domains in alpha-subunit of a HERGchannel (Blaustein and Miller, 2004) is employed. To improve performanceof the fusion, a peptide connector may be introduced between DhaA andthe N- and/or C-terminal fragments of the loop.

In yet another embodiment, when fused to a protein expressed on the cellsurface, a mutant hydrolase on the cell surface, when combined with aligand of the invention, e.g., one which contains a fluorophore, may beemployed to monitor internalization of membrane protein. If ligand ofinvention is microenvironment sensitive, the system may be employed tomonitor changes of environment surrounding membrane protein. In oneembodiment, the ligand of the invention is one that has low or nopermeability to the cell membrane. In one embodiment, labeling of DhaAexpressed on cell surface with non-permeant ligand followed by treatmentof the cells with cell permeant ligand, can be used to monitorsimultaneously relocation of surface and internal pool of membraneprotein. Alternatively, such a system can be used to monitor the effectof different agents, e.g., drugs, on different pools of membraneproteins.

In yet another embodiment, when fused to a protein expressed on the cellsurface, a mutant hydrolase on the cell surface, when combined with aligand of the invention, e.g., one which contains a detectablefunctional group, may be employed to monitor modification of themembrane proteins (e.g., proteolysis, glycosylation, etc.).Alternatively, such a system can be used to monitor the effect ofdifferent agents, e.g., drugs, on modification of the membrane proteins.

In yet another embodiment, when fused to an ion channel, a mutanthydrolase on the cell surface, when combined with a ligand of theinvention, e.g., one which contains a microenvironmental sensitivefunctional group, may be employed to monitor functional activity of thechannel. Alternatively, such a system can be used to monitor the effectof different agents (and/or conditions), e.g., drugs (and/or a change oftemperature, stretching of cell membrane, interaction of the cells withsolid surfaces, other cells, proteins) on ion channel activity.

REFERENCES

-   Ambler et al., Biochem. J., 276:4710 (1991).-   Arshady et al., Macromol. Chem., 187:687 (1981).-   Ausubel et al., Current Protocols in Molecular Biology, Vol. III,    A.1(3-4), Supplement 38 (1997).-   Blaustein and Miller, Nature, 427: 499-500 (2004).-   Boshart et al., Cell, 41:521 (1985).-   Bosma et al., Appl. Environ. Microbial., 68:3582 (2002).-   Breitinger and Becker, ChemBioChem, 3:1042 (2002).-   Chalfie, M. and Kain, S. R., eds., GFP: Green Fluorescent Protein    Strategies and Applications (Wiley, New York, 1998).-   Cornish-Bowden, in Fundamentals of Enzyme Kinetics, pp 1-17,    Portland Press Ltd., London (1995).-   Cubitt et al., Trends Biochem. Sci., 20:448 (1995).-   Danen & Yamada, J. Cell Physiology, 189:1 (2001).-   De Angelis et al., Proc. Natl. Acad. Sci. USA, 95:12312 (1998).-   Ed Harlow, David Lane, In: Antibodies: A Laboratory Manual, Cold    Spring Harbor Laboratory Press, p. 726 (1988)-   Eu and Andrade, Luminescence, 16:57-63 (2001).-   Farinas et al., J. Biol. Chem., 274:7603 (1999).-   Franken et al., EMBO J., 10:1297 (1991).-   Gardiner-Garden et al., J. Mol. Biol., 196:261 (1987).-   Gorman et al., Proc Natl Acad Sci USA, 79:6777 (1982).-   Griffin et al., Science, 281:269 (1998).-   Hatta and Takeichi, Nature, 320:447 (1986).-   Hatta et al., J Cell Biol., 106:873 (1998).-   Hermanson, Bioconjugate Techniques, Academic Press, San Diego,    Calif. (1996).-   Hiscox et al., BBRC, 293:714 (2002).-   Ho et al., Gene, 77:51 (1989).-   Hohesnester & Engel, Matrix Biology, 21:115 (2002).-   Holloway et al., J. Microbiol. Methods, 32:31 (1998).-   Hynkova et al., FEBS Lett., 446:177 (1999).-   Janssen et al., Eur. J. Biochem., 171:67 (1988).-   Janssen et al., J. Bacteriol., 171:6791 (1989).-   Jarvik and Telmer, Ann. Rev. Genet., 32:601-618 (1998).-   Keppler et al., Nature Biotechnology, 21:86 (2003).-   Keuning et al., J. Bacteriol., 163:635 (1985).-   Kim et al., Gene, 91:217 (1990).-   Kinoshita and Inoue, Curr. Opin. Chem. Biol., 4: 632 (2000).-   Kneen et al., Biophys. J., 74:1591 (1998).-   Krooshof et al., Biochemistry, 36:9571 (1997).-   Kulakova et al., Microbiology, 143:109 (1997).-   Kwon et al., Anal. Chem., 76:5713 (2004).-   Lakowicz, J. R. Principles of Fluorescence Spectroscopy, New York:    Plenum Press (1983).-   Llopis et al., Proc. Natl. Acad. Sci. USA, 95:6803 (1998).-   Medof and Tykocinski, In: Welply J K, Jaworski E, editors. In:    Glycobiology. New York: Wiley-Liss, p. 17-22 (1990)-   Miesenböck et al., Nature, 394:192 (1998).-   Minasov et al., J. Am. Chem. Soc., 124:5333 (2002).-   Miyawaki et al., Nature, 388:882 (1967).-   Mizushima and Nagata, Nucleic Acids Res., 18:5322 (1990).-   Murray et al., Nucleic Acids Res., 17:477 (1989).-   Nagata et al., Appl. Environ. Microbiol., 63:3707 (1997).-   Nakamura et al., Nucl. Acids. Res., 28:292 (2000).-   Newman et al., Biochemistry, 38, 16105 (1999).-   Nollet et al., J Mol Biol, 299:551 (2000).-   Ormö et al., Science, 273:1392 (1996).-   Pieters et al., Bioorg. & Medicinal Chem. Lett., 9:161 (1999).-   Pries et al., Biochemistry, 33:1242 (1994).-   Pries et al., J. Biol. Chem., 270:10405 (1995).-   Qureshi et al., J. Biol. Chem., 276:46422 (2001).-   Ragaut et al., Nat. Biotechnol., 17:1030-1032 (1999).-   Rosomer et al., J. Biol. Chem., 272:13270 (1997).-   Sallis et al., J. Gen. Microbiol., 136:115 (1990).-   Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. 2001.-   Sanger et al., Proc. Natl. Acad. Sci. U.S.A., 74:5463 (1977).-   Savage et al., Avidin-Biotin Chemistry: A Handbook (Pierce Chemical    Company, Rockford, Ill.) (1992).-   Schindler, Biochemistry, 38:5772 (1999).-   Scholtz et al., J. Bacteriol., 169:5016 (1987).-   Sevelever et al., In: Young N S, Moss J, editors. Paroxysmal    Nocturnal Hemoglobinuria and the Glycophosphoinositol-Linked    Proteins”. San-Diego: CA: Academic Press, p 199 (2000).-   Siegel and Isacoff, Neuron, 19:735 (1997).-   Silverman, Mechanism-based enzyme in activation, in Methods    Enzymology, 249:240 (1995).-   Stevens, Biochem. J., 310: 361 (1995).-   Stroffekova et al., Eur. J. Physiol., 442:859 (2001).-   Takeichi et al., Dev Biol., 87:340 (1981).-   Tiede et al., J. Biol. Chem., 380:503 (1999).-   Tsien, Ann. Rev. Biochem., 67:509 (1998).-   Udenfriend and Kodukula, Meth. Enzymol., 250:571 (1995).-   Uetsuki et al., J Biol. Chem., 264:5791 (1989).-   Wada et al., Nucleic Acids Res., 18 Suppl:2367 (1990).-   Yokota et al., J. Bacteriol., 169:4049 (1987).-   Zawadzke et al., Protein Engineering, 8:1275 (1995).-   Zlokarnik et al., Science, 279:84 (1998).

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

What is claimed is:
 1. A fusion polypeptide of a protein of interest anda mutant haloalkane dehalogenase having at least 85% sequence identityto the wild type dehalogenase polypeptide of SEQ ID NO:82 and comprisingone to three amino acid substitutions at positions 106, 130, and 272relative to the wild-type dehalogenase.
 2. The fusion polypeptide ofclaim 1, wherein the protein of interest is a selectable marker protein,membrane protein, cytosolic protein, nuclear protein, structuralprotein, an enzyme, an enzyme substrate, a receptor protein, atransporter protein, a transcription factor, a channel protein, aphospho-protein, a kinase, a signaling protein, a metabolic protein, amitochondrial protein, a receptor associated protein, a nucleic acidbinding protein, an extracellular matrix protein, a secreted protein, areceptor ligand, a serum protein, an immunogenic protein, a fluorescentprotein, or a protein with reactive cysteine.
 3. The fusion polypeptideof claim 1, wherein said mutant haloalkane dehalogenase is covalentlyattached to a substrate.
 4. The fusion polypeptide of claim 3, whereinthe substrate comprises a functional group.
 5. The fusion polypeptide ofclaim 4, wherein the functional group is a molecule with one or moreproperties that facilitate detection or isolation of the substrate withthe mutant haloalkane dehalogenase attached covalently thereto.
 6. Thefusion polypeptide of claim 4, wherein the functional group is selectedfrom the group consisting of a fluorophore, chromophore, luminophore,nucleic acid, a peptide, a solid support, a radionuclide, a contrastagent, a metal ion chelator, an affinity molecule, a drug, a toxin, asubstrate for an enzyme, an inhibitor of an enzyme, a protein ligand, aDNA intercalator, and a crosslinker.
 7. The fusion polypeptide of claim1, wherein the substitution relative to the wild type dehalogenasepolypeptide is one substitution at amino acid position
 272. 8. Thefusion polypeptide of claim 7, wherein the amino acid at position 272 isphenylalanine.
 9. The fusion polypeptide of claim 1, further comprisingsubstitutions relative to the wild type dehalogenase polypeptide at oneto three of amino acid positions 175, 176 and 273.